Technetium-99 conjugated with methylene diphosphonate (99Tc-MDP) is a novel bisphosphonate derivative without radioactivity and has been successfully used to treat arthritis in China for years. Since bisphosphonate therapy has the potential to induce bisphosphonate-associated osteonecrosis of the jaw (BRONJ), we examine whether 99Tc-MDP represents a new class of bisphosphonate for anti-resorptive therapy to ameliorate estrogen deficiency–induced bone resorption with less risk of causing BRONJ. We showed that 99Tc-MDP-treated ovariectomized (OVX) mice had significantly improved bone mineral density (BMD) and trabecular bone volume in comparison to the untreated OVX group by inhibiting osteoclasts and enhancing osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). To determine the potential of inducing BRONJ, 99Tc-MDP/dexamethasone (Dex) or zoledronate/Dex were administered into C57BL/6J mice via the tail vein, followed by extraction of maxillary first molars. Interestingly, 99Tc-MDP treatment showed less risk to induce osteonecrosis in the maxillary bones compared to zoledronate treatment group, partially because 99Tc-MDP neither suppressed adaptive regulatory T cells (Tregs) nor activated the inflammatory T-helper-producing interleukin 17 cells (Th17). Taken together, our findings demonstrate that 99Tc-MDP therapy may be a promising approach in the treatment of osteoporosis with less risk of causing BRONJ.
99Tc-MDP; bisphosphonate-related osteonecrosis of the jaw; ovariectomized mice; bone mineral density; bone marrow mesenchymal stem cells
Vascular calcification, prevalent in diabetes and chronic kidney disease, contributes to morbidity and mortality. To investigate the effect of receptor activator of NF-kB ligand (RANKL) on vascular calcification in vivo, transgenic mice, where RANKL expression was targeted to vascular smooth muscle cells using SM22alpha promoter (SM22α-Rankltg), was created. Sixteen-month-old male SM22α-Rankltg mice had higher body weight and higher serum calcium levels but lower lumbar bone mineral density (BMD) compared with age- and gender-matched WT littermates. BMD of long bones, body fat (% of weight) of the leg, and serum levels of phosphate and RANKL were not significantly different. No significant differences in these parameters were observed in the female mice. Histological analysis did not reveal calcium deposits in the aortic roots of the SM22α-Rankltg mice. To analyze the osteoblastic differentiation and mineralization potentials of vascular cells, aortic smooth muscle cells (SMCs) were isolated and cultured. Results showed that SM22α-Rankltg SMCs had higher baseline alkaline phosphatase (ALP) activity, but not the baseline matrix calcification. When induced by the PKA agonist, forskolin, ALP activity was greater in SM22α-Rankltg than in WT SMCs. Realtime RT-qPCR revealed higher baseline expression of ALP and ankylosis genes, but lower osteoprotegerin gene, in SM22α-Rankltg SMCs. Matrix mineralization induced by inorganic phosphate or forskolin was greater in SM22α-Rankltg than in WT SMCs. Treatment of these cells with the ALP inhibitor, levamisole, abolished forskolin-induced matrix mineralization but not Pi-induced matrix mineralization. These findings suggest that RANKL overexpression in the vasculature may promote mineralization potential.
RANKL; smooth muscle cells; vascular; calcification; SM22 alpha
The extent to which nutrient intake may influence bone structure and strength during maximum rates of skeletal growth remains uncertain.
To examine the relationship of dietary intake of micronutrients and bone macro-architectural structure in young girls.
This cross-sectional analysis included baseline data from 363 4th and 6th grade girls enrolled in the Jump-In study. Nutrient intake was assessed using the Harvard Youth/Adolescent Food Frequency Questionnaire. Volumetric BMD (vBMD), bone geometry and strength were measured by peripheral quantitative computed tomography (pQCT). Correlations and regression modeling assessed relations between usual nutrient intake and bone parameters.
In 4th grade girls, metaphyseal and diaphyseal area and circumferences, and diaphyseal strength were associated with vitamin C intake (r = 0.15–0.19; p<0.05). Zinc intake was correlated with diaphyseal vBMD (r = 0.15–0.16; p<0.05). Using multiple linear regression to adjust for important covariates, we observed significant independent associations for vitamin C and zinc with bone parameters. For every mg/d of vitamin C intake trabecular area increased by 11%, cortical strength improved by 14%; and periosteal and endosteal circumferences increased by 5% and 8.6%, respectively. For every mg/d of zinc intake, cortical vBMD increased by <1%. No significant associations were observed in 6th-grade girls.
Results of this study suggests that vitamin C and zinc intake are positively associated with objective measures of bone geometry, size and strength in 4th-grade girls. This indicates potential differences in micronutrient and bone associations at various age-associated stages of bone maturation perhaps indicative of competing hormonal influences.
peripheral quantitative computed tomography (pQCT); preadolescence; bone mineral density (BMD); bone strength; micronutrient intake; vitamin C
Relationships between geological phosphorite deposition and biological apatite nucleation have often been overlooked. However, similarities in biological apatite and phosphorite mineralogy suggest that their chemical formation mechanisms may be similar. This review serves to draw parallels between two newly described phosphorite mineralization processes, and proposes a similar novel mechanism for biologically controlled apatite mineral nucleation. This mechanism integrates polyphosphate biochemistry with crystal nucleation theory. Recently, the roles of polyphosphates in the nucleation of marine phosphorites were discovered. Marine bacteria and diatoms have been shown to store and concentrate inorganic phosphate (Pi) as amorphous, polyphosphate granules. Subsequent release of these P reserves into the local marine environment as Pi results in biologically induced phosphorite nucleation. Pi storage and release through an intracellular polyphosphate intermediate may also occur in mineralizing oral bacteria. Polyphosphates may be associated with biologically controlled apatite nucleation within vertebrates and invertebrates. Historically, biological apatite nucleation has been attributed to either a biochemical increase in local Pi concentration or matrix-mediated apatite nucleation control. This review proposes a mechanism that integrates both theories. Intracellular and extracellular amorphous granules, rich in both calcium and phosphorus, have been observed in apatite-biomineralizing vertebrates, protists, and atremate brachiopods. These granules may represent stores of calcium-polyphosphate. Not unlike phosphorite nucleation by bacteria and diatoms, polyphosphate depolymerization to Pi would be controlled by phosphatase activity. Enzymatic polyphosphate depolymerization would increase apatite saturation to the level required for mineral nucleation, while matrix proteins would simultaneously control the progression of new biological apatite formation.
Biomineralization: mechanisms; Bone and cartilage development; Crystal structure/crystallinity
The main function of osteoclasts in vivo is the resorption of bone matrix, leaving behind typical resorption traces consisting of pits and trails. The mechanism of pit formation is well described, but less is known about trail formation. Pit-forming osteoclasts possess round actin rings. In this study we show that trail-forming osteoclasts have crescent-shaped actin rings and provide a model that describes the detailed mechanism. To generate a trail, the actin ring of the resorption organelle attaches with one side outside the existing trail margin. The other side of the ring attaches to the wall inside the trail, thus sealing that narrow part to be resorbed next (3–21 μm). This 3D configuration allows vertical resorption layer-by-layer from the surface to a depth in combination with horizontal cell movement. Thus, trails are not just traces of a horizontal translation of osteoclasts during resorption. Additionally, we compared osteoclastic resorption on bone and dentin since the latter is the most frequently used in vitro model and data are extrapolated to bone. Histomorphometric analyses revealed a material-dependent effect reflected by an 11-fold higher resorption area and a sevenfold higher number of pits per square centimeter on dentin compared to bone. An important material-independent aspect was reflected by comparable mean pit area (μm2) and podosome patterns. Hence, dentin promotes the generation of resorbing osteoclasts, but once resorption has started, it proceeds independently of material properties. Thus, dentin is a suitable model substrate for data acquisition as long as osteoclast generation is not part of the analyses.
Osteoclast; Bone; Dentin; Resorption model; Trail formation
Parathyroid hormone (PTH) is the most effective osteoporosis treatment, but it is only effective if administered by daily injections. We fused PTH(1–33) to a collagen binding domain (PTH–CBD) to extend its activity, and have shown an anabolic bone effect with monthly dosing. We tested the duration of action of this compound with different routes of administration. Normal young C57BL/6J mice received a single intraperitoneal injection of PTH–CBD (320 μg/kg). PTH–CBD treated mice showed a 22.2 % increase in bone mineral density (BMD) at 6 months and 12.8 % increase at 12 months. When administered by subcutaneous injection, PTH–CBD again caused increases in BMD, 15.2 % at 6 months and 14.3 % at 12 months. Radiolabeled PTH–CBD was concentrated in bone and skin after either route of administration. We further investigated skin effects of PTH–CBD, and histological analysis revealed an apparent increase in anagen VI hair follicles. A single dose of PTH–CBD caused sustained increases in BMD by >10 % for 1 year in normal mice, regardless of the route of administration, thus showing promise as a potential osteoporosis therapy.
Animal models; Bone density technology; DXA; Osteoporosis therapy
Deletion of connexin (Cx) 43 from osteoblasts and osteocytes (OCN-Cre;Cx43fl/− mice) or from osteocytes only (DMP1-8kb-Cre;Cx43fl/fl mice) results in increased cortical, but not cancellous, osteocyte apoptosis and widening of the femoral midshaft without changes in cortical thickness. Despite the consequent larger moment of inertia, stiffness and ultimate load, measures of mechanical strength assessed by 3-point bending, are not higher in either model of Cx43 deficiency, due to reduced Young's modulus, a measure of the stiffness of the material per unit of area. In OCN-Cre;Cx43fl/− mice, this was accompanied by a reduced ratio of nonreducible/reducible collagen cross-links as assessed by Fourier Transformed Infrared Imaging (FTIRI) in the femoral diaphysis. On the other hand, DMP1-8kb-Cre;Cx43fl/fl mice did not show a significant reduction in collagen maturation in the same skeletal site, but a small decrease in mineralization was detected by FTIRI. Remarkably, both osteoblastic and osteocytic cells lacking Cx43 expressed lower mRNA levels of lysyl oxidase, a crucial enzyme involved in collagen maturation. These findings suggest that Cx43 expression in osteoblasts is involved in maintaining the quality of the bone matrix in cortical bone through the maturation of collagen cross-links. Osteocytic Cx43 expression is important also to maintain the stiffness of the bone material, where a Cx43 deficiency results in a local reduction in mineralization, possibly due to osteocyte apoptosis.
connexin 43; young's modulus; FTIR; osteocyte; osteoblast; Lox
During endochondral bone formation, chondrocytes and osteoblasts synthesize and mineralize the extracellular matrix through a process that initiates within matrix vesicles (MVs) and ends with bone mineral propagation onto the collagenous scaffold. pH gradients have been identified in the growth plate of long bones, but how pH changes affect the initiation of skeletal mineralization is not known. Tissue-nonspecific alkaline phosphatase (TNAP) degrades extracellular inorganic pyrophosphate (ePPi), a mineralization inhibitor produced by ectonucleotide pyrophosphatase/ phosphodiesterase-1 (NPP1), while contributing Pi from ATP to initiate mineralization. TNAP and NPP1, alone or combined, were reconstituted in dipalmitoylphosphatidylcholine (DPPC) liposomes to mimic the microenvironment of MVs. The hydrolysis of ATP, ADP, AMP and PPi was studied at pH 8 and 9 and compared to the data determined at pH 7.4. While catalytic efficiencies in general were higher at alkaline pH, PPi hydrolysis was maximal at pH 8 and indicated a preferential utilization of PPi over ATP, at pH 8 versus 9. In addition, all proteoliposomes induced mineral formation when incubated in a synthetic cartilage lymph (SCL) containing 1 mM ATP as substrate and amorphous calcium phosphate (ACP) or calciumphosphate- phosphatidylserine complexes (PS-CPLX) as nucleators. Propagation of mineralization was significantly more efficient at pHs 7.5 and 8 than at pH 9. Since a slight pH elevation from 7.4 to 8 promotes considerably more hydrolysis of ATP, ADP and AMP primarily by TNAP, this small pH change facilitates mineralization, especially via upregulated PPi hydrolysis by both NPP1 and TNAP, further elevating the Pi/PPi ratio, thus enhancing bone mineralization.
biomineralization; alkaline pH; microenvironment; proteoliposomes; pyrophosphate; ATP
18F-fluoride positron emission tomography (18F-PET) allows the assessment of regional bone formation and could have a role in the diagnosis of adynamic bone disease (ABD) in patients with chronic kidney disease (CKD). The purpose of this study was to examine bone formation at multiple sites of the skeleton in hemodialysis patients (CKD5D) and assess the correlation with bone biopsy. Seven CKD5D patients with suspected ABD and 12 osteoporotic postmenopausal women underwent an 18F-PET scan, and bone plasma clearance, Ki, was measured at ten skeletal regions of interest (ROI). Fifteen subjects had a transiliac bone biopsy following double tetracycline labeling. Two CKD5D patients had ABD confirmed by biopsy. There was significant heterogeneity in Ki between skeletal sites, ranging from 0.008 at the forearm to 0.028 mL/min/mL at the spine in the CKD5D group. There were no significant differences in Ki between the two study groups or between the two subjects with ABD and the other CKD5D subjects at any skeletal ROI. Five biopsies from the CKD5D patients had single tetracycline labels only, including the two with ABD. Using an imputed value of 0.3 μm/day for mineral apposition rate (MAR) for biopsies with single labels, no significant correlations were observed between lumbar spine Ki corrected for BMAD (Ki/BMAD) and bone formation rate (BFR/BS), or MAR. When biopsies with single labels were excluded, a significant correlation was observed between Ki/BMAD and MAR (r = 0.81, p = 0.008) but not BFR/BS. Further studies are required to establish the sensitivity of 18F-PET as a diagnostic tool for identifying CKD patients with ABD.
Adynamic bone disease; 18F-fluoride PET; Chronic kidney disease; Osteoporosis; Bone histomorphometry; Bone formation
Intracortical microstructure influences crack propagation and arrest within bone cortex. Genetic variation in intracortical remodeling may contribute to mechanical integrity and, therefore, fracture risk. Our aim was to determine the degree to which normal population-level variation in intracortical microstructure is due to genetic variation. We examined right femurs from 101 baboons (74 females, 27 males; aged 7–33 years) from a single, extended pedigree to determine osteon number, osteon area (On.Ar), haversian canal area, osteon population density, percent osteonal bone (%On.B), wall thickness (W.Th), and cortical porosity (Ct.Po). Through evaluation of the covariance in intracortical properties between pairs of relatives, we quantified the contribution of additive genetic effects (heritability [h2]) to variation in these traits using a variance decomposition approach. Significant age and sex effects account for 9 % (Ct.Po) to 21 % (W.Th) of intracortical microstructural variation. After accounting for age and sex, significant genetic effects are evident for On.Ar (h2 = 0.79, p = 0.002), %On.B (h2 = 0.82, p = 0.003), and W.Th (h2 = 0.61, p = 0.013), indicating that 61–82 % of the residual variation (after accounting for age and sex effects) is due to additive genetic effects. This corresponds to 48–75 % of the total phenotypic variance. Our results demonstrate that normal, population-level variation in cortical microstructure is significantly influenced by genes. As a critical mediator of crack behavior in bone cortex, intracortical microstructural variation provides another mechanism through which genetic variation may affect fracture risk.
Primate; Osteoporosis; Biomechanics; Population studies; Bone histomorphometry
Thiazolidinediones (TZDs), peroxisome proliferator-activated receptor gamma activators, and insulin sensitizers represent drugs used to treat hyperglycemia in diabetic patients. Type 2 diabetes mellitus (T2DM) is associated with a twofold increase in fracture risk, and TZDs use increases this risk by an additional twofold. In this study, we analyzed the effect of systemic administration of the TZD rosiglitazone on new bone formation in two in vivo models of bone repair, a model of drilled bone defect regeneration (BDR) and distraction osteogenesis (DO) and a model of extended bone formation. Rosiglitazone significantly inhibited new endosteal bone formation in both models. This effect was correlated with a significant accumulation of fat cells, specifically at sites of bone regeneration. The diminished bone regeneration in the DO model in rosiglitazone-treated animals was associated with a significant decrease in cell proliferation measured by the number of cells expressing proliferating cell nuclear antigen and neovascularization measured by both the number of vascular sinusoids and the number of cells producing proangiogenic vascular endothelial growth factor at the DO site. In summary, rosiglitazone decreased new bone formation in both BDR and DO models of bone repair by mechanisms which include both intrinsic changes in mesenchymal stem cell proliferation and differentiation and changes in the local environment supporting angiogenesis and new bone formation. These studies suggest that bone regeneration may be significantly compromised in T2DM patients on TZD therapy.
Rosiglitazone; Diabetes; Bone healing; Osteoblast; Angiogenesis; Adipocyte
The skeletal system functions as a locomotive organ and a mineral reservoir and combinations of genetic and environmental factors affect the skeletal system. Although delayed puberty is associated with compromised bone mass, suppression of estrogen should be beneficial to cortical strength. The purpose was to employ path analysis to study bone strength and delayed puberty. Forty-five female rats were randomly assigned to a control group (n = 15) and an experimental group (n = 30) that received injections of gonadotropin releasing hormone antagonist (GnRH-a). Causal models were constructed by specifying directed paths between bone traits. The first model tested the hypothesis that the functional relationships between bone traits and body weight were altered by a delay in pubertal onset. GnRH-a injections during puberty altered the covariation between body weight and bone size. The second model was constructed to test the hypothesis that variability in stiffness was causally related to variability in body weight. The model also tested the relationship between the periosteal and endocortical surfaces and their relationship to stiffness. There was no change in the relationship between the surfaces in the GnRH-a group. The third model determined the effect of estradiol on both total area and relative cortical area in both groups. The relationship between periosteal surface and serum estradiol levels was only significant during estrogen suppression. These data suggest that increases in body weight during or prior to puberty may not be protective of bone strength.
Bone architecture/structure; Biomechanics; Bone histology; Bone histomorphometry; Steroid hormones; Estrogens; Path analysis
The incidence of menstrual irregularities, both primary and secondary amenorrhea, has been reported to be as high as 60%, with the highest incidence in younger athletes, suggesting possible adverse effects on bone development. It was hypothesized that in a rat model, suppressed hypothalamic activity via a gonadotropin-releasing hormone antagonist (GnRH-a) before onset of puberty would result in a relatively larger bone strength deficit compared with suppression after puberty. Hypothalamic suppression was achieved by providing GnRH injections. Animals received injections for 25 days either before puberty (pre group) (age 23–46 days) or after puberty (post group) (age 65–90 days). Body weights and uterine weights were measured. Serum estradiol was assayed. Mechanical strength of the right femora and histomorphometry of the left femur were measured. Suppression of the hypothalamic– pituitary–gonadal axis was confirmed by significant atrophy of uterine tissue and suppressed estradiol levels. The peak moment was significantly lower in the pre and post GnRH-a groups compared with control. The percentage difference of the average peak moment and stiffness values from the respective age-matched control groups yielded a greater percentage difference in the pre group. The cortical area was less in the GnRH-a-treated groups, but no significant difference between the relative deficits between pre and post groups were found. Hypothalimic–pituitary–gonadal axis suppression before puberty resulted in a significantly larger deficit in mechanical strength compared with postpubertal animals. The time before puberty may represent a time when skeletal strength is more compromised. Women experience both primary and secondary amenorrhea; however, the treatment may need to be different for each condition.
Puberty; Estradiol; Bone strength; GnRH antagonist; Bone histomorphometry
Calcium plus vitamin D (CaD) supplementation has a modest but significant effect on slowing loss of femoral bone mass and reducing risk of hip fractures in adherent postmenopausal women. The goal of this study was to determine if CaD supplementation influences hip structural parameters that are associated with fracture risk.
We studied 1,970 postmenopausal women enrolled in the Women’s Health Initiative randomized controlled trial of CaD at one of three bone mineral density (BMD) clinical centers. Hip structural analysis software measured BMD and strength parameters on DXA scans at three regions: femoral narrow neck, intertrochanteric and shaft. Random effects models were used to test the average differences in hip BMD and geometry between intervention and placebo.
There was greater preservation of hip BMD at the narrow neck with CaD relative to placebo across six years of intervention. CaD also altered the underlying cross-sectional geometry at the narrow neck in the direction of greater strength with small increases in cross-sectional area and section modulus, and a decrease in buckling ratio with CaD relative to placebo. While trends at both the intertrochanter and shaft regions were similar to those noted at the narrow neck, no significant intervention effects were evident. There was no significant interaction of CaD and age or baseline calcium levels for hip structural properties.
CaD supplementation is associated with modest beneficial effects on hip structural features at the narrow neck, which may explain some of the benefit of CaD in reducing hip fracture risk.
Calcium and Vitamin D Supplementation; Bone Strength; Postmenopausal Women; Hip Structure
The drawbacks of estrogen restrict the clinical use of hormone replacement therapy, and it would be most helpful to explore new estrogenic substances that could prevent bone loss and be free from any adverse effects. We synthesized a new compound named bone-seeking estrogen (SE2) by combining 17β-estradiol (E2) with iminodiacetic acid through the Mannich reaction. E2 and SE2 were labeled with isotope 3H, and the tissue distribution tests of E2-3H and SE2-3H were analyzed by the radioactivity. The specific nuclear binding of E2 and SE2 in osteoblasts was measured. SE2 exhibited significantly greater affinity for bone but lower affinity for ovary and uterus than did E2, and SE2 maintained a high affinity for the estrogen receptor alpha similar to that of E2. SE2 administration did not induce uterine hypertrophy. Body weight increase was significantly suppressed by treatment with E2 but not by SE2 after ovariectomy (OVX). SE2 decreased bone turnover as E2 after OVX detected by serum biochemical markers. Bone histology and micro-CT analysis revealed that SE2 administration, similar to E2, could improve bone mass and trabecular architecture after OVX. Biomechanical analyses showed that SE2 treatment effectively increased mechanical properties after OVX. The results suggested that SE2 was effective in preventing OVX-induced bone loss and exhibited few side effects on body weight and uterine hypertrophy, which was beneficial in reducing the adverse effects caused by E2. SE2 may be a better choice than E2 for the prevention of postmenopausal osteoporosis.
Bone-seeking estrogen; Estrogen; Postmenopausal osteoporosis; Side effects; Trabecular architecture
Bone formation in a variety of contexts depends on angiogenesis, however there are few reports of the vascular response to osteogenic skeletal loading. We used the rat forelimb compression model to characterize vascular changes after fatigue loading. The right forelimbs of 72 adult rats were loaded cyclically in vivo to one of four displacement levels, to produce four discrete levels of ulnar damage. Rats were euthanized 3–14 days after loading and their vasculature perfused with silicone rubber. Transverse histological sections were cut along the ulnar diaphysis. We quantified vessel number, average vessel area, total vessel area and bone area. On day 3 we observed a dramatic periosteal expansion near the ulnar midshaft, with significant increases in periosteal vascularity; total vessel area was increased 250–450% (p<0.001). Vascularity remained elevated on days 7 and 14. Vessel number and average vessel area were not correlated (p=0.09) and contributed independently to total vascular increases. Bone area was not increased on day 3, but on days 7 and 14 was increased significantly in all displacement groups (p<0.01) due to periosteal woven bone formation. Vascular and bone changes depended on longitudinal location (p<0.001), with peak increases 2 mm distal to the midshaft. Vascular and bone changes also depended on displacement level (p<0.005), with greater increases at higher levels of fatigue displacement. We conclude that skeletal fatigue loading induces a rapid increase in periosteal vascularity, followed by an increase in bone area. The angiogenic-osteogenic response is spatially coordinated and scaled to the level of the mechanical stimulus.
This randomized, double-blind study assessed the antifracture efficacy and safety of intermittent intravenous (IV) ibandronate versus oral daily risedronate in Japanese patients with primary osteoporosis. Ambulatory patients aged ≥60 years were randomized to receive 0.5 or 1 mg/month IV ibandronate plus oral daily placebo or 2.5 mg/day oral risedronate, the licensed dose in Japan, plus IV placebo. The primary end point was noninferiority of ibandronate versus risedronate for first new or worsening vertebral fracture over 3 years. A total of 1,265 patients were randomized. A total of 1,134 patients formed the per-protocol set. Both ibandronate doses were noninferior to risedronate: 0.5 mg, hazard ratio (HR) 1.09 [95 % confidence interval (CI) 0.77–1.54]; 1 mg, HR 0.88 (95 % CI 0.61–1.27). The rate of first new vertebral fracture over 3 years was 16.8 % (95 % CI 12.8–20.8) for 0.5 mg ibandronate, 11.6 % (95 % CI 8.2–15.0) for 1 mg ibandronate, and 13.2 % (95 % CI 9.6–16.9) for risedronate. Significant increases in bone mineral density relative to baseline were observed with all treatments after 6 months, with substantial reductions in bone turnover markers after 3 months. Greatest efficacy was obtained with 1 mg ibandronate. Analyses in women only showed similar results to the overall population. No new safety concerns were identified. This study demonstrated the noninferiority of IV ibandronate to the licensed Japanese dose of oral risedronate and suggested that 1 mg/month is an effective dose in Japanese patients with primary osteoporosis.
Ibandronate; Intravenous; Osteoporosis; Risedronate; Vertebral fracture
We report two immuoreactive species of thrombospondin-2 (TSP2), sized approximately 200 and 125 kDa, in the long bones of growing, but not skeletally mature, mice. In vitro osteoblasts secrete a 200-kDa species into the culture medium as early as day 3, and it appears in the cell-matrix layer by day 7. A 125-kDa species appears in the cell-matrix layer in parallel with mineralization; it is not detected in cell-conditioned medium. Unilateral tibial fracture induced a time-dependent upregulation of the 200-kDa species at the site of trauma. By contrast, relative levels of the 125-kDa species at the fracture site were lower than in bones from naive control animals. In the contralateral untouched control tibia, the 200-kDa species was rapidly and substantially reduced compared to bone harvested from naive control mice. Levels of the 125-kDa species in the untouched tibia declined gradually with time postfracture. TSP2 gene expression in uninjured control bone decreased modestly by 21 days postfracture. On the day of fracture, the osteoblast differentiation potential of MSCs harvested from uninjured bones decreased compared to those harvested from naive control animals. The presence of two isoforms suggests that TSP2 may undergo posttranscriptional or posttranslational processing in skeletal tissue. Our data also suggest that, in the context of trauma, the two TSP2 isforms are differentially modulated at injured and noninjured skeletal sites in an animal undergoing fracture healing.
Matricellular; Thrombospondin-2; Fracture; Marrow stromal cell; Osteoblast
Our previous work has shown associations between childhood adiposity and perinatal methylation status of several genes in umbilical cord tissue, including endothelial nitric oxide synthase (eNOS). There is increasing evidence that eNOS is important in bone metabolism; we therefore related the methylation status of the eNOS gene promoter in stored umbilical cord to childhood bone size and density in a group of 9-year old children.
We used Sequenom MassARRAY to assess the methylation status of 2 CpGs in the eNOS promoter, identified from our previous study, in stored umbilical cords of 66 children who formed part of a Southampton birth cohort and who had measurements of bone size and density at age 9 years (Lunar DPXL DXA instrument).
Percentage methylation varied greatly between subjects. For one of the two CpGs, eNOS chr7:150315553+, after taking account of age and sex there was a strong positive association between methylation status and the child’s whole body bone area (r=0.28,p=0.02), bone mineral content (r=0.34,p=0.005) and areal bone mineral density (r=0.34,p=0.005) at age 9 years. These associations were independent of previously documented maternal determinants of offspring bone mass.
Our findings suggest an association between methylation status at birth of a specific CpG within the eNOS promoter and bone mineral content in childhood. This supports a role for eNOS in bone growth and metabolism and implies that its contribution may at least in part occur during early skeletal development.
Epigenetic; methylation; umbilical cord; eNOS; DXA
There is increasing interest in identifying new pathways and candidate genes that confer susceptibility to osteoporosis. There is evidence that adipogenesis and osteogenesis may be related, including a common bone marrow progenitor cell for both adipocytes and osteoblasts. Perilipin 1 (PLIN1) and Perilipin 4 (PLIN4) are members of the PATS family of genes and are involved in lipolysis of intracellular lipid deposits. A previous study reported gender-specific associations between one polymorphism of PLIN1 and bone mineral density (BMD) in a Japanese population. We hypothesized that polymorphisms in PLIN1 and PLIN4 would be associated with bone measures in adult Caucasian participants of the Framingham Osteoporosis Study (FOS). We genotyped 1,206 male and 1,445 female participants of the FOS for four single-nucleotide polymorphism (SNPs) in PLIN1 and seven SNPs in PLIN4 and tested for associations with measures of BMD, bone ultrasound, hip geometry, and height. We found several gender-specific significant associations with the measured traits. The association of PLIN4 SNP rs8887, G>A with height in females trended toward significance after simulation testing (adjusted P = 0.07) and remained significant after simulation testing in the combined-sex model (adjusted P = 0.033). In a large study sample of men and women, we found a significant association between one SNP in PLIN4 and height but not with bone traits, suggesting that PATS family genes are not important in the regulation of bone. Identification of genes that influence human height may lead to a better understanding of the processes involved in growth and development.
Perilipin 1; Perilipin 4; Bone mineral density; Bone geometry; Framingham Osteoporosis Study
We review the various aspects of health technology assessment in osteoporosis, including epidemiology and burden of disease, and assessment of the cost-effectiveness of recent advances in the treatment of osteoporosis and the prevention of fracture, in the context of the allocation of health-care resources by decision makers in osteoporosis. This article was prepared on the basis of a symposium held by the Belgian Bone Club and the discussions surrounding that meeting and is based on a review and critical appraisal of the literature. Epidemiological studies confirm the immense burden of osteoporotic fractures for patients and society, with lifetime risks of any fracture of the hip, spine, and forearm of around 40 % for women and 13 % for men. The economic impact is also large; for example, Europe’s six largest countries spent €31 billion on osteoporotic fractures in 2010. Moreover, the burden is expected to increase in the future with demographic changes and increasing life expectancy. Recent advances in the management of osteoporosis include novel treatments, better fracture-risk assessment notably via fracture risk algorithms, and improved adherence to medication. Economic evaluation can inform decision makers in health care on the cost-effectiveness of the various interventions. Cost-effectiveness analyses suggest that the recent advances in the prevention and treatment of osteoporosis may constitute an efficient basis for the allocation of scarce health-care resources. In summary, health technology assessment is increasingly used in the field of osteoporosis and could be very useful to help decision makers efficiently allocate health-care resources.
Burden of disease; Cost-effectiveness; Economic evaluation; Health technology assessment; Osteoporosis
Mechanical loading induces positive changes in the skeleton due to direct effects on bone cells, which may include regulation of transcription factors that support osteoblast differentiation and function. Flow effects on osteoblast transcription factors have generally been evaluated after short exposures. In this work, we assayed flow effects on osteogenic genes at early and late time points in a preosteoblast (CIMC-4) cell line and evaluated both steady and oscillatory flows. Four hours of steady unidirectional flow decreased the level of RANKL mRNA 53 ± 7% below that of nonflowed cells, but increases in Runx2 and osterix mRNA (44 ± 22% and 129 ± 12%, respectively) were significant only after 12–19 h of continuous flow. Late flow effects on RANKL and osterix were also induced by an intermittent flow–rest protocol (four cycles of 1 h on/1 h off + overnight rest). Four hours of oscillatory flow decreased RANKL mRNA at this early time point (63 ± 2%) but did not alter either osterix or Runx2. When oscillatory flow was delivered using the intermittent flow–rest protocol, Runx2 and osterix mRNA increased significantly (85 ± 19% and 161 ± 22%, respectively). Both β-catenin and ERK1/2, known to be involved in RANKL regulation, were rapidly activated by steady flow. Inhibition of flow-activated ERK1/2 prevented the increase in osterix mRNA but not Runx2; Runx2 phosphorylation was increased by flow, an effect which likely contributes to osterix induction. This work shows that both steady and oscillatory fluid flows can support enhancement of an osteogenic phenotype.
Loading; Mechanical; Osteoblast; Shear; ERK1/2
Bone loss during perimenopause, an estrogen-sufficient period, correlates with elevated serum follicle-stimulating hormone (FSH) and decreased inhibins A and B. Utilizing a recently described ovotoxin-induced animal model of perimenopause characterized by a prolonged estrogen-replete period of elevated FSH, we examined longitudinal changes in bone mineral density (BMD) and their association with FSH. Additionally, serum inhibin levels were assessed to determine whether elevated FSH occurred secondary to decreased ovarian inhibin production and, if so, whether inhibins also correlated with BMD. BMD of the distal femur was assessed using dual-energy X-ray absorptiometry (DXA) over 19 months in Sprague-Dawley rats treated at 1 month with vehicle or 4-vinylcyclohexene diepoxide (VCD, 80 or 160 mg/kg daily). Serum FSH, inhibins A and B, and 17-ß estradiol (E2) were assayed and estrus cyclicity was assessed. VCD caused dose-dependent increases in FSH that exceeded values occurring with natural senescence, hastening the onset and prolonging the duration of persistent estrus, an acyclic but E2-replete period. VCD decreased serum inhibins A and B, which were inversely correlated with FSH (r2 = 0.30 and 0.12, respectively). In VCD rats, significant decreases in BMD (5–13%) occurred during periods of increased FSH and decreased inhibins, while BMD was unchanged in controls. In skeletally mature rats, FSH (r2 = 0.13) and inhibin A (r2 = 0.15) correlated with BMD, while inhibin B and E2 did not. Thus, for the first time, both the hormonal milieu of perimenopause and the association of dynamic perimenopausal changes in FSH and inhibin A with decreased BMD have been reproduced in an animal model.
4-Vinylcyclohexene diepoxide; Follicle-stimulating hormone; Inhibin; Bone; Estradiol; Osteoporosis; Perimenopause
Numerous whole-body vibration (WBV) devices of various forces are available on the market, although their influence on the musculoskeletal system is not yet understood. The effect of different WBVs on bone healing and muscle function was evaluated in rats ovariectomized at 3 months of age. 2 months after ovariectomy, bilateral metaphyseal tibia osteotomy and T-plate osteosynthesis were performed. Rats were divided into groups: intact, OVX, and OVX exposed to vertical WBVs of 35, 50, 70, or 90 Hz (experiment 1) or horizontal WBVs of 30, 50, 70, or 90 Hz (experiment 2) 5 days after osteotomy (0.5 mm, 15 min/day for 30 days). The tibia and gastrocnemius and soleus muscles were collected. Vertical vibrations (>35 Hz) improved cortical and callus densities, enlarged callus area and width, suppressed the tartrate-resistant acid phosphatase gene, enhanced citrate synthase activity, accelerated osteotomy bridging (35 and 50 Hz), upregulated the osteocalcin (Oc) gene (70 Hz), and increased relative muscle weight (50 Hz). Horizontal vibrations reduced cortical width (<90 Hz) and callus density (30 Hz), enhanced alkaline phosphatase (Alp) gene expression (50 Hz), decreased the size of oxidative fibers (35 and 70 Hz), and increased capillary density (70, 90 Hz). Biomechanical data; serum Oc, Alp, and creatine kinase activities; body weight; and food intake did not change after WBVs. Vertical WBVs of 35 and 50 Hz produced more favorable results than the higher frequencies. Horizontal WBV showed no positive or negative effects. Further studies are needed to elucidate the effects of WBV on different physiological systems, and precautions must be taken when implementing WBV in the treatment of patients.
Whole-body vibration; Osteoporosis; Bone healing; Muscle; Ovariectomized rat