Pipes Output http://pipes.yahoo.com/pipes/pipe.info?_id=9f9d8b24eee6f920ac6285f05e0d9c73 Thu, 01 Oct 2015 23:14:15 +0000 http://pipes.yahoo.com/pipes/ http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2726 Claudin-16 protein (CLDN16) is a component of tight junctions (TJ) with a restrictive distribution so far demonstrated mainly in the kidney. Here, we demonstrate the expression of CLDN16 also in the tooth germ and show that claudin-16 gene (CLDN16) mutations results in Amelogenesis imperfecta (AI) in the five studied patients with Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis (FHHNC). To investigate the role of CLDN16 in tooth formation, we studied a murine model of FHHNC and showed that CLDN16 deficiency led to altered secretory ameloblast TJ structure, to lowering of extracellular pH in the forming enamel matrix, and to abnormal enamel matrix protein processing, resulting in an enamel phenotype closely resembling human AI. This study unravels an association of FHHNC due to CLDN16 mutations with AI which is directly related to the loss of function of CLDN16 during amelogenesis. Overall, this study indicates for the first time the importance of a TJ protein in tooth formation and underlines the need to establish a specific dental follow-up for these patients. This article is protected by copyright. All rights reserved Thu, 01 Oct 2015 16:02:43 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2724 Introduction Evidence suggests that serotonin is an inhibitor of bone formation. We aimed to assess i) serum serotonin levels in patients with ankylosing spondylitis (AS), a prototype bone forming disease, compared to patients with rheumatoid arthritis (RA) and healthy subjects ii) the effect(s) of TNFα blockers on serum serotonin levels in patients with AS and RA and iii) the effect(s) of serum of AS patients on serotonin signaling. Methods Serum serotonin levels were measured in 47 patients with AS, 28 patients with RA and 40 healthy subjects by RIA; t-test was used to assess differences between groups. The effect of serum on serotonin signaling was assessed using the human osteoblastic cell line Saos2, evaluating levels of phospho-CREB by western immunoblots. Results Serotonin serum levels were significantly lower in patients with AS compared to healthy subjects (mean ± SEM ng/ml: 122.9 ± 11.6 vs 177.4 ± 24.58, p = 0.038) and patients with RA (mean ± SEM ng/ml: 244.8 ± 37.5, p = 0.0004). Patients with AS receiving TNFα blockers had significantly lower serotonin levels compared to patients with AS not on such treatment (mean ± SEM ng/ml: 95.8 ± 14.9 vs 149.2 ± 16.0, p = 0.019). Serotonin serum levels were inversely correlated with pCREB induction in osteoblast-like Saos-2 cells. Conclusions Serotonin levels are low in patients with AS and decrease even further during anti-TNFα treatment. Differences in serotonin levels are shown to have a functional impact on osteoblast-like Saos-2 cells. Therefore, serotonin may be involved in new bone formation in AS. This article is protected by copyright. All rights reserved Wed, 30 Sep 2015 05:50:28 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2723 While the role of ERα in regulating bone metabolism has been extensively studied, ERβ has been largely dismissed as a relevant modulator of bone mass. Previous studies examining ERβ utilized a germline knock-out mouse expressing transcript variants of ERβ and displaying systemic hormonal changes that confounded interpretation of the skeletal phenotype. Thus, we used a conditional ERβ mouse model to achieve deletion of ERβ specifically in early osteoprogenitor cells using the Prx1-Cre driver. We observed marked increases in the trabecular bone volume fraction (of 58% [P < 0.003] and 93% [P < 0.0003] in 6- and 12-week old female ERβPrx1-CKO mice, respectively) but no changes in cortical bone. Serum estradiol and IGF-I levels were unaltered in ERβPrx1-CKO mice. Bone formation and resorption indices by histomorphometry and serum assays were unchanged in these mice, suggesting that alterations in bone turnover may have occurred early in development. However, the ratio of colony-forming unit-osteoblasts (CFU-OBs) to CFU-fibroblasts (CFU-Fs) was increased in bone marrow cultures from ERβPrx1-CKO compared to control mice, indicating increased differentiation of osteoblast precursor cells into osteoblasts in ERβPrx1-CKO mice. Detailed quantitative polymerase chain reaction analyses of 128 genes in 16 pre-specified pathways revealed significant downregulation of 11 pathways in ERβPrx1-CKO mice. Thus, deletion of ERβ specifically in osteoblast lineage cells, in the absence of all splice variants, increases trabecular bone mass and modulates multiple pathways related to bone metabolism. These findings suggest that pharmacological inhibition of ERβ in bone may provide a novel approach to treat osteoporosis. This article is protected by copyright. All rights reserved Tue, 29 Sep 2015 07:08:06 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2722 Hypersecretion of acid hydrolases is a hallmark feature of mucolipidosis II (MLII), a lysosomal storage disease caused by loss of carbohydrate-dependent lysosomal targeting. Inappropriate extracellular action of these hydrolases is proposed to contribute to skeletal pathogenesis but the mechanisms that connect hydrolase activity to the onset of disease phenotypes remain poorly understood. Here we link extracellular cathepsin K activity to abnormal bone and cartilage development in MLII animals by demonstrating that it disrupts the balance of TGFß-related signaling during chondrogenesis. TGFß-like Smad2,3 signals are elevated and BMP-like Smad1,5,8 signals reduced in both feline and zebrafish MLII chondrocytes and osteoblasts, maintaining these cells in an immature state. Reducing either cathepsin K activity or expression of the transcriptional regulator Sox9a in MLII zebrafish significantly improved phenotypes. We further identify components of the large latent TGFß complex as novel targets of cathepsin K at neutral pH, providing a possible mechanism for enhanced Smad2,3 activation in vivo. These findings highlight the complexity of the skeletal disease associated with MLII and bring new insight to the role of secreted cathepsin proteases in cartilage development and growth factor regulation. This article is protected by copyright. All rights reserved Fri, 25 Sep 2015 03:48:19 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2720 Bone homeostasis is maintained by the synergistic actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Although interferon-related developmental regulator 1 (Ifrd1) has been identified as a transcriptional coactivator/repressor in various cells, little attention has been paid to its role in osteoblastogenesis and bone homeostasis thus far. Here, we show that Ifrd1 is a critical mediator of both the cell-autonomous regulation of osteoblastogenesis and osteoblast-dependent regulation of osteoclastogenesis. Osteoblast-specific deletion of murine Ifrd1 increased bone formation and decreased bone resorption, causing high bone mass. Ifrd1 deficiency enhanced osteoblast differentiation and maturation along with increased expression of Runx2 and Osterix (Osx). Mechanistically, Ifrd1 deficiency increased the acetylation status of p65, a component of NF-κB, at residues K122 and K123 via the attenuation of the interaction between p65 and histone deacetylase (HDAC). This led to the nuclear export of p65 and a decrease in NF-κB-dependent Smad7 expression and the subsequent enhancement of Smad1/Smad5/Smad8-dependent transcription. Moreover, a high bone mass phenotype in the osteoblast-specific deletion of Ifrd1 was markedly rescued by the introduction of one Osx-floxed allele but not of Runx2-floxed allele. Coculture experiments revealed that Ifrd1-deficient osteoblasts have a higher osteoprotegerin (Opg) expression and a lower ability to support osteoclastogenesis. Ifrd1 deficiency attenuated the interaction between β-catenin and HDAC, subsequently increasing the acetylation of β-catenin at K49, leading to its nuclear accumulation and the activation of the β-catenin-dependent transcription of Opg. Collectively, the expression of Ifrd1 in osteoblasts repressed osteoblastogenesis and activated osteoclastogenesis through modulating the NF-κB/Smad/Osx and β-catenin/OPG pathways, respectively. These findings suggest that Ifrd1 has a pivotal role in bone homeostasis through its expression in osteoblasts in vivo and represents a therapeutic target for bone diseases. This article is protected by copyright. All rights reserved Tue, 22 Sep 2015 07:06:11 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2721 Milk fat globule-epidermal growth factor 8 (MFG-E8) is an anti-inflammatory glycoprotein that mediates the clearance of apoptotic cells and is implicated in the pathogenesis of autoimmune and inflammatory diseases. As MFG-E8 also controls bone metabolism, we investigated its role in rheumatoid arthritis (RA) focusing on inflammation and joint destruction. The regulation of MFG-E8 by inflammation was assessed in vitro using osteoblasts, in arthritic mice and in patients with RA. K/BxN serum transfer arthritis (STA) was applied to MFG-E8 knock-out mice to assess its role in the pathogenesis of arthritis. Stimulation of osteoblasts with lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α down-regulated the expression of MFG-E8 by 30-35%. MFG-E8-deficient osteoblasts responded to LPS with a stronger production of pro-inflammatory cytokines. In vivo, MFG-E8 mRNA levels were 52% lower in the paws of collagen-induced arthritic (CIA) mice and 24-42% lower in the serum of arthritic mice using two different arthritis models (CIA and STA). Similarly, patients with RA (n = 93) had lower serum concentrations of MFG-E8 (-17%) as compared to healthy controls (n = 140). In a subgroup of patients who had a moderate to high disease activity (n = 21) serum concentrations of MFG-E8 rose after complete or partial remission had been achieved (+67%). Finally, MFG-E8-deficient mice subjected to STA exhibited a stronger disease burden, an increased number of neutrophils in the joints, and a more extensive local and systemic bone loss. This was accompanied by an increased activation of osteoclasts and a suppression of osteoblast function in MFG-E8-deficient mice. Thus, MFG-E8 is a protective factor in the pathogenesis of RA and subsequent bone loss. Whether MFG-E8 qualifies as a novel biomarker or therapeutic target for the treatment of RA is worth addressing in further studies. This article is protected by copyright. All rights reserved Tue, 22 Sep 2015 07:05:39 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2717 Cells with in vitro properties similar to those of bone marrow stromal stem cells are present in tooth pulp as quiescent cells that are mobilised by damage. These dental pulp stem cells (DPSCs) respond to damage by stimulating proliferation and differentiation into odontoblast-like cells that form dentine to repair the damage. In continuously growing mouse incisors, tissue at the incisor tips is continuously being damaged by the shearing action between the upper and lower teeth acting to self-sharpen the tips. We investigated mouse incisor tips as a model for the role of DPSCs in a continuous natural repair/regeneration process. We show that the pulp at the incisor tip is composed of a disorganised mass of mineralised tissue produced by odontoblast-like cells. These cells become embedded into the mineralised tissue that is rapidly formed and then lost during feeding. Tetracycline labelling revealed the expected incorporation into newly synthesised dentine formation of the incisor, but also a zone covering the pulp cavity at the tips of the incisors that is mineralised very rapidly. This tissue was dentine-like, but had a significantly lower mineral content than dentine as determined by Raman spectroscopy. The mineral was more crystalline than dentine, indicative of small, defect-free mineral particles. To identify the origin of cells responsible for deposition of mineralised tissue we genetically labelled perivascular cells by crossing NG2ERT2Cre and Nestin Cre mice with reporter mice. A large number of pericyte-derived cells were visible in the pulp of incisor tips with some having elongated, odontoblast-like shapes. These results show that in mouse incisors, rapid, continuous mineralisation occurs at the tip to seal-off the pulp tissue from the external environment. The mineral is formed by perivascular-derived cells that differentiate into cells expressing DSPP and produce a dentine-like material in a process that functions as continuous natural tissue regeneration. This article is protected by copyright. All rights reserved Tue, 22 Sep 2015 07:03:13 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2718 Previous prospective cohort studies have shown that serum levels of sex steroids and sex hormone-binding globulin (SHBG) associate with non-vertebral fracture risk in men. The predictive value of sex hormones and SHBG for vertebral fracture risk specifically is, however, less studied. Elderly men (aged ≥65 years) from Sweden and Hong Kong participating in the MrOS study had baseline estradiol and testosterone analyzed by GC-MS and SHBG by IRMA. Incident clinical vertebral fractures (n = 242 cases) were evaluated in 4324 men during an average follow-up of 9.1 years. In a subsample of these men (n = 2256), spine X-rays were obtained at baseline and after an average follow-up of 4.3 years to identify incident radiographic vertebral fractures (n = 157 cases). The likelihood of incident clinical and radiographic vertebral fractures was estimated by Cox proportional hazards models and logistic regression models, respectively. Neither serum estradiol (HR per SD increase, 95% CI: 0.93, 0.80-1.08) nor testosterone (1.05, 0.91-1.21) predicted incident clinical vertebral fractures in age-adjusted models in the combined data set. High serum SHBG, however, associated with increased clinical vertebral fracture risk (1.24, 1.12-1.37). This association remained significant after further adjustment for FRAX® with or without BMD. SHBG also associated with increased incident radiographic vertebral fracture risk (combined data set; OR per SD increase, 95% CI: 1.23, 1.05-1.44). This association remained significant after adjustment for FRAX® with or without BMD. In conclusion, high SHBG predicts incident clinical and radiographic vertebral fractures in elderly men and adds moderate information beyond FRAX® with BMD for vertebral fracture risk prediction. This article is protected by copyright. All rights reserved Tue, 22 Sep 2015 07:01:27 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2719 Cathepsin K (CK), a lysosomal cysteine protease is highly expressed in mature osteoclasts and degrades type 1 collagen. Odanacatib (ODN) is a selective and reversible CK inhibitor that inhibits bone loss in preclinical and clinical studies. Although an anti-resorptive, ODN, does not suppress bone formation which led us to hypothesize that ODN may display restorative effect on theosteopenic bones. In a curative study, skeletally mature New Zealand rabbits were ovarectomized (OVX) and after induction of bone loss were given a steady-state exposure of ODN (9μM/day) for 14 weeks. Sham operated and OVX rabbits treated with alendronate (ALD), 17β-estradiol (E2) or PTH served as various controls. Efficacy was evaluated by assessing BMD, bone microarchitecture (using microcomputed tomography), fluorescent labeling of bone, and biomechanical strength. Skeletal Ca/P ratio was measured by scanning electron microscopy (SEM) with X-ray microanalysis, crystallinity by X-ray diffraction, and bone mineral density distribution (tissue mineralization) by backscattered SEM. Between the sham and ODN-treated osteopenic groups, lumbar and femur metaphyseal BMD, Ca/P ratio, trabecular microstructure and geometric indices, vertebral compressive strength, trabecular lining cells, cortical parameters (femoral area and thickness, and periosteal deposition) and serum P1NP were largely comparable. Skeletal improvements in ALD-treated or E2-treated groups fell significantly short of the sham/ODN/PTH group. However, the ODN group displayed reduced ductility and enhanced brittleness of central femur, which might have been contributed by higher crytallinity and tissue mineralization. Rabbit bone marrow stromal cells expressed CK and when treated with ODN displayed increased formation of mineralized nodules and decreased apoptosis in serum-deficient medium compared with control. In vivo, ODN did not suppress remodeling but inhibited osteoclast activity more than ALD. Taken together, we show that ODN reverses BMD, skeletal architecture and compressive strength in osteopenic rabbits; however, it increases crystallinity and tissue mineralization, thus leading to increased cortical bone brittleness. This article is protected by copyright. All rights reserved Tue, 22 Sep 2015 06:59:42 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2715 The aim of this case study is to describe changes in areal bone mineral density (aBMD) by dual-energy X-ray absorptiometry (DXA) scan as well as volumetric bone density and microarchitecture by high-resolution peripheral quantitative computed tomography (HRpQCT) in two patients with autosomal dominant osteopetrosis (ADO) and compare to 20 healthy subjects. We describe a 44-years-old male patient with six low impact fractures since he was sixteen, and a 32-years-old female patient with four low impact fractures on her past history. Radiographic changes were typical of ADO. Consistent with the much higher aBMD, total volumetric BMD (average bone density of the whole bone, including trabecular and cortical compartments) at distal radius and tibia (HRpQCT) was more than twice the mean values found in healthy subjects, in both patients. Trabecular number and thickness were higher, leading to an evident increase in trabecular bone volume to tissue volume. Also, an enormous increase in cortical thickness was found. Most important, a great heterogeneity in bone microstructure of the affected patients was evident on HRpQCT images: islets of very dense bone were interposed with areas with apparent normal density. The increase in aBMD, vBMD and most indices of trabecular and cortical bone, associated with the great heterogeneity on bone tridimensional microarchitecture, reflect the accumulation of old and fragile bone randomly distributed along the skeleton. These alterations in bone microstructure probably compromise bone quality, which might justify the high prevalence of low impact fractures in patients with ADO, despite abnormally elevated BMD. This article is protected by copyright. All rights reserved Mon, 21 Sep 2015 06:56:27 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2713 Trabecular Bone Score (TBS) has been shown to predict major osteoporotic (clinical vertebral, hip, humerus, and wrist) and hip fractures in post-menopausal women and older men, but the association of TBS with these incident fractures in men independent of prevalent radiographic vertebral fracture is unknown. TBS was estimated on AP spine DXA scans obtained at the baseline visit for 5,979 men age ≥65 years enrolled in MrOS and its association with incident major osteoporotic and hip fractures estimated with proportional hazards models. Model discrimination was tested with Harrell's C-statistic and with a categorical net reclassification improvement index, using 10-year risk cutpoints of 20% for major osteoporotic and 3% for hip fractures. For each standard deviation decrease in TBS, there were hazard ratios of 1.27 (95% C.I. 1.17 to 1.39) for major osteoporotic fracture, and 1.20 (95% C.I. 1.05 to 1.39) for hip fracture, adjusted for FRAX with BMD 10 year fracture risks and prevalent radiographic vertebral fracture. In the same model, those with prevalent radiographic vertebral fracture compared to those without prevalent radiographic vertebral fracture had hazard ratios of 1.92 (95% C.I. 1.49 to 2.48) for major osteoporotic fracture and 1.86 (95% C.I. 1.26 to 2.74) for hip fracture. There were improvements of 3.3%, 5.2%, and 6.2%, respectively, of classification of major osteoporotic fracture cases when TBS, prevalent radiographic vertebral fracture status, or both were added to FRAX with BMD and age, with minimal loss of correct classification of non-cases. Neither TBS nor prevalent radiographic vertebral fracture improved discrimination of hip fracture cases or non-cases. In conclusion, TBS and prevalent radiographic vertebral fracture are associated with incident major osteoporotic fractures in older men independent of each other and FRAX 10 year fracture risks, and these data support their use in conjunction with FRAX for fracture risk assessment in older men. This article is protected by copyright. All rights reserved Thu, 17 Sep 2015 07:26:15 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2712 WNT/β-CATENIN signaling is involved in multiple aspects of skeletal development including chondrocyte differentiation and maturation. While the functions of β-CATENIN in chondrocytes have been extensively investigated through gain-of-function and loss-of-function mouse models, the precise downstream effectors through which β-CATENIN regulates these processes are not well defined. Here, we report that the matricellular protein, CCN1, is induced by WNT/β-CATENIN signaling in chondrocytes. Specifically, we found that β-CATENIN signaling promotes CCN1 expression in isolated primary sternal chondrocytes and both embryonic and postnatal cartilage. Additionally, we show that, in vitro, CCN1 overexpression promotes chondrocyte maturation while inhibition of endogenous CCN1 function inhibits maturation. To explore the role of CCN1 on cartilage development and homeostasis in vivo, we generated a novel transgenic mouse model for conditional Ccn1 overexpression and show that cartilage-specific CCN1 overexpression leads to chondrodysplasia during development and cartilage degeneration in adult mice. Finally, we demonstrate that CCN1 expression increases in mouse knee joint tissues following meniscal/ligamentous injury (MLI) and in human cartilage following meniscal tear. Collectively, our data suggest that CCN1 is an important regulator of chondrocyte maturation during cartilage development and homeostasis. This article is protected by copyright. All rights reserved Sat, 12 Sep 2015 09:37:36 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2711 Regulation of gene expression changes during chondrogenic differentiation by DNA methylation and demethylation is little understood. Methylated cytosines (5mC) are oxidized by the ten-eleven-translocation (TET) proteins to 5-hydroxymethylcytosines (5hmC), 5-formylcytosines (5fC) and 5-carboxylcytosines (5caC) eventually leading to a replacement by unmethylated cytosines (C) i.e. DNA demethylation. Additionally, 5hmC is stable and acts as an epigenetic mark by itself. Here, we report that global changes in 5hmC mark chondrogenic differentiation in vivo and in vitro. Tibia anlagen and growth plate analyses during limb development at mouse embryonic days E 11.5, 13.5 and 17.5 showed dynamic changes in 5hmC levels in the differentiating chondrocytes. A similar increase in 5hmC levels was observed in the ATDC5 chondroprogenitor cell line accompanied by increased expression of the TET proteins during in vitro differentiation. Loss of TET1 in ATDC5 decreased 5hmC levels and impaired differentiation, demonstrating a functional role for TET1-mediated 5hmC dynamics in chondrogenic differentiation. Global analyses of the 5hmC-enriched sequences during early and late chondrogenic differentiation identified 5hmC distribution to be enriched in the regulatory regions of genes preceding the transcription start site (TSS) as well as in the gene bodies. Stable gains in 5hmC were observed in specific subsets of genes including genes associated with cartilage development and in chondrogenic lineage-specific genes. 5hmC gains in regulatory promoter and enhancer regions as well as in gene bodies were strongly associated with activated but not repressed genes, indicating a potential regulatory role for DNA hydroxymethylation in chondrogenic gene expression. This article is protected by copyright. All rights reserved Sat, 12 Sep 2015 09:37:14 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2710 Aseptic loosening and periprosthetic infection leading to inflammatory osteolysis is a major complication associated with total joint arthroplasty (TJA). The liberation of bacterial products and/or implant-derived wear particles activates immune cells which produce pro-osteoclastogenic cytokines that enhances osteoclast recruitment and activity leading to bone destruction and osteolysis. Therefore agents which prevent the inflammatory response and/or attenuate excessive osteoclast (OC) formation and bone resorption offer therapeutic potential by prolonging the life of TJA implants. Alexidine dihydrochloride (AD) is a bisbiguanide compound commonly used as an oral disinfectant and in contact lens solutions. It possesses anti-microbial, anti-inflammatory and anti-cancer properties however its effects on OC biology are poorly described. Here, we demonstrate that AD inhibits OC formation and bone resorption in vitro and exert prophylatic protection against LPS-induced osteolysis in vivo. Biochemical analysis demonstrated that AD suppressed RANKL-induced activation of MAPKs (ERK, p38 and JNK) leading to the downregulation of NFATc1. Furthermore, AD disrupted F-actin ring formation and attenuated the ability of mature OC to resorb bone. Collectively our findings suggest that AD may be a promising prophylactic anti-osteoclastic/resorptive agent for the treatment of osteolytic diseases caused by excessive OC formation and function. This article is protected by copyright. All rights reserved Sat, 12 Sep 2015 09:31:19 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2709 Vitamin C is an important antioxidant and cofactor which is involved in the regulation of development, function and maintenance of several cell types in the body. Deficiencies in vitamin C can lead to conditions such as scurvy, which, among other ailments, causes gingivia, bone pain and impaired wound healing. This review examines the functional importance of vitamin C as it relates to the development and maintenance of bone tissues. Analysis of several epidemiological studies and genetic mouse models regarding the effect of vitamin C shows a positive effect on bone health. Overall, vitamin C exerts a positive effect on trabecular bone formation by influencing expression of bone matrix genes in osteoblasts. Recent studies on the molecular pathway for vitamin C actions that include direct effects of vitamin C on transcriptional regulation of target genes by influencing the activity of transcription factors and by epigenetic modification of key genes involved in skeletal development and maintenance are discussed. With an understanding of mechanisms involved in the uptake and metabolism of vitamin C and knowledge of precise molecular pathways for vitamin C actions in bone cells, it is possible that novel therapeutic strategies can be developed or existing therapies can be modified for the treatment of osteoporotic fractures. This article is protected by copyright. All rights reserved Fri, 11 Sep 2015 04:23:51 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2708 Bisphosphonates (BPs) are the most commonly used medications for osteoporosis. This ASBMR report provides guidance on BP therapy duration with a risk benefit perspective. Two trials provided evidence for long-term BP use. In the Fracture Intervention Trial Long-term Extension (FLEX), postmenopausal women receiving alendronate for 10 years had fewer clinical vertebral fractures than those switched to placebo after 5 years. In the HORIZON extension, women who received 6 annual infusions of zoledronic acid had fewer morphometric vertebral fractures compared with those switched to placebo after 3 years. Low hip T-score, between -2 and -2.5 in FLEX and below -2.5 in HORIZON extension, predicted a beneficial response to continued therapy. Hence, the Task Force suggests that after 5 years of oral BP or 3 years of intravenous BP, reassessment of risk should be considered. In women at high risk, for example, older women, those with a low hip T-score or high fracture risk score, those with previous major osteoporotic fracture, or who fracture on therapy, continuation of treatment for up to 10 years (oral) or 6 years (intravenous), with periodic evaluation, should be considered. The risk of atypical femoral fracture, but not osteonecrosis of the jaw, clearly increases with BP therapy duration, but such rare events are outweighed by vertebral fracture risk reduction in high risk patients. For women not at high fracture risk after 3-5 years of BP treatment, a drug holiday of 2-3 years can be considered. The suggested approach for long term BP use is based on limited evidence, only for vertebral fracture reduction, in mostly Caucasian postmenopausal women, and does not replace the need for clinical judgment. It may be applicable to men and patients with glucocorticoid-induced osteoporosis, with some adaptations. It is unlikely that future trials will provide data for formulating definitive recommendations. This article is protected by copyright. All rights reserved Wed, 09 Sep 2015 01:29:29 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2706 High bone mass (HBM) can be an incidental clinical finding; however, monogenic HBM disorders (e.g. LRP5 or SOST mutations) are rare. We aimed to determine to what extent HBM is explained by mutations in known HBM genes. 258 unrelated HBM cases were identified from review of 335,115 DXA scans from 13 UK centers. Cases were assessed clinically and underwent sequencing of known anabolic HBM loci: LRP5 (exons 2, 3, 4), LRP4 (exons 25, 26), SOST (exons 1, 2, and the van Buchem's disease (VBD) 52kb intronic deletion 3'). Family members were assessed for HBM segregation with identified variants. Three-dimensional protein models were constructed for identified variants. Two novel missense LRP5 HBM mutations ([c.518C > T; p.Thr173Met], [c.796C > T; p.Arg266Cys]) were identified, plus three previously reported missense LRP5 mutations ([c.593A > G; p.Asn198Ser], [c.724G > A; p.Ala242Thr], [c.266A > G; p.Gln89Arg]), associated with HBM in 11 adults from seven families. Individuals with LRP5 HBM (∼prevalence 5/100,000) displayed a variable phenotype of skeletal dysplasia with increased trabecular BMD and cortical thickness on HRpQCT, and gynoid fat mass accumulation on DXA, compared with both non-LRP5 HBM and controls. One mostly asymptomatic woman carried a novel heterozygous nonsense SOST mutation (c.530C > A; p.Ser177X) predicted to prematurely truncate sclerostin. Protein modelling suggests the severity of the LRP5-HBM phenotype corresponds to the degree of protein disruption and the consequent effect on SOST-LRP5 binding. We predict p.Asn198Ser and p.Ala242Thr directly disrupt SOST binding; both correspond to severe HBM phenotypes (BMD Z-scores +3.1 to +12.2, inability to float). Less disruptive structural alterations predicted from p.Arg266Cys, p.Thr173Met, p.Gln89Arg were associated with less severe phenotypes (Z-scores +2.4 to +6.2, ability to float). In conclusion, although mutations in known HBM loci may be asymptomatic, they only account for a very small proportion (∼3%) of HBM individuals, suggesting the great majority are explained by either unknown monogenic causes or polygenic inheritance. This article is protected by copyright. All rights reserved Tue, 08 Sep 2015 07:36:25 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2705 Mineralized collagen fibrils are composed of tropocollagen molecules and mineral crystals derived from hydroxyapatite to form a composite material that combines optimal properties of both constituents and exhibits incredible strength and toughness. Their complex hierarchical structure allows collagen fibrils to sustain large deformation without breaking. In this study, we report a mesoscale model of a single mineralized collagen fibril using a bottom-up approach. By conserving the three-dimensional structure and the entanglement of the molecules, we are able to construct finite size fibril models that allow us to explore the deformation mechanisms that govern their mechanical behavior under large deformation. We investigate the tensile behavior of a single collagen fibril with various intrafibrillar mineral content and find that a mineralized collagen fibril can present up to five different deformation mechanisms to dissipate energy. These mechanisms include molecular uncoiling, molecular stretching, mineral/collagen sliding, molecular slippage and crystal dissociation. By multiplying its sources of energy dissipation and deformation mechanisms, a collagen fibril can reach impressive strength and toughness. Adding mineral into the collagen fibril can increase its strength up to 10 times and its toughness up to 35 times. Combining cross-links with mineral make the fibril stiffer but more brittle. We also find that a mineralized fibril reaches its maximum toughness to density and strength to density ratios for a mineral density of around 30%. This result, in good agreement with experimental observations, attests that bone tissue is optimized mechanically to remain lightweight but maintain strength and toughness. This article is protected by copyright. All rights reserved Mon, 07 Sep 2015 05:45:28 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2702 Adynamic bone is the most frequent type of bone lesion in patients with chronic kidney disease and long-term use of anti-resorptive therapy may also lead to the adynamic bone condition. The hallmark of adynamic bone is a loss of bone turnover and a major clinical concern of adynamic bone is diminished bone quality and an increase in fracture risk. Our current study aims to investigate how bone quality changes with age in our previously established mouse model of adynamic bone. Young and old mice (4- and 16-months) were used in this study. Col2.3Δtk (DTK) mice were treated with ganciclovir and pamidronate to create the adynamic bone condition. Bone quality was evaluated using established techniques including bone histomorphometry, microcomputed tomography, quantitative backscattered electron imaging, and biomechanical testing. Changes in mineral and matrix properties were examined by powder x-ray diffraction and Raman spectroscopy. Ageing Controls had a natural decline in bone formation and resorption with a corresponding deterioration in trabecular bone structure. Bone turnover was severely blunted at all ages in Adynamic animals which preserved trabecular bone loss normally associated with ageing. However, the preservation of trabecular bone mass and structure in old Adynamic mice did not rescue deterioration of bone mechanical properties. There was also a decrease in cortical bone toughness in old Adynamic mice that was accompanied by a more mature collagen matrix and longer bone crystals. Little is known about the effects of metabolic bone disease on bone fracture resistance. We observed an age-related decrease in bone toughness that was worsened by the adynamic condition, and this decrease may be due to material level changes at the tissue level. Our mouse model may be useful in the investigation of the mechanisms involved in fractures occurring in elderly patients on anti-resorptive therapy who have very low bone turnover. This article is protected by copyright. All rights reserved Wed, 02 Sep 2015 17:09:37 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2698 Receptor activator of nuclear factor-κB ligand (RANKL) is a TNF-like cytokine that is necessary for osteoclast formation and survival. Elevated RANKL synthesis is associated with both increased osteoclast number and bone resorption. Earlier studies identified an enhancer 76 kb upstream of the Tnfsf11 transcriptional start site (TSS) termed RL-D5 or the distal control region (DCR) that modulates RANKL expression in response to PTH, 1,25(OH)2D3, and an array of cytokines. Mice lacking RL-D5 exhibit high bone mass associated with decreased RANKL expression in bone, spleen, and thymus. In addition to RL-D5, genome-wide studies have identified 9 additional Tnfsf11 enhancers residing upstream of the gene's TSS, which provide RANKL cell type-specificity and responsiveness to local and systemic factors. ChIP-chip analysis has revealed inducible VDR and CREB binding at an enhancer termed RL-D2 23 kb upstream of the Tnfsf11 TSS in osteoblastic ST2 cells. Herein, we use ChIP-seq analysis to confirm this finding and delete this enhancer from the mouse genome to determine its physiological role in vivo. RL-D2-/- primary stromal cells showed decreased RANKL-induction by both forskolin and 1,25(OH)2D3 ex vivo. Consistent with this, the PTH induction of RANKL expression was significantly blunted in RL-D2-/- mice in vivo. In contrast, lack of RL-D2 had no effect on 1,25(OH)2D3 induction of RANKL in vivo. Similar to the results seen in RL-D5-/- mice, lack of RL-D2 led to decreased skeletal RANKL expression, resulting in decreased osteoclast numbers and a progressive increase in bone mineral density. Lack of RL-D2 increased cancellous bone mass in femur and spine, but did not alter femoral cortical bone thickness. These results highlight the role of distal enhancers in the regulation of RANKL expression by PTH and perhaps 1,25(OH)2D3, and suggest that the RL-D2 and RL-D5 enhancers contribute in either an additive or synergistic manner to regulate bone remodeling. This article is protected by copyright. All rights reserved Mon, 31 Aug 2015 18:21:58 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2605 In contrast to traditional approaches to fracture risk assessment using clinical risk factors and Bone Mineral Density (BMD), a new technique, Reference Point micro-Indentation (RPI), permits direct assessment of bone quality; in vivo tibial RPI measurements appear to discriminate patients with a fragility fracture from controls. However, it is unclear how this relates to the site of the most clinically devastating fracture, the femoral neck, and whether RPI provides information complementary to that from existing assessments. Femoral neck samples were collected at surgery following low trauma hip fracture (n = 46; 17 male, 83 (IQR 77-87) years), and compared, using RPI (Biodent HfcTM), with 16 cadaveric control samples, free from bone disease (7 male; 65 (IQR 61-74) years). A subset of fracture patients returned for Dual-energy X-ray Absorptiometry (DXA) assessment (Hologic Discovery) and, for the controls, a micro-computed tomography setup (HMX, Nikon) was used to replicate DXA scans. The indentation depth was greater in femoral neck samples from osteoporotic fracture patients than controls (p < 0.001), which persisted with adjustment for age, sex, BMI and height (p < 0.001) but was site-dependent, being less pronounced in the inferomedial region. RPI demonstrated good discrimination between fracture and controls using ROC analysis (AUC = 0.79 to 0.89), and a model combining RPI to clinical risk factors or BMD performed better than the individual components (AUC = 0.88 to 0.99). In conclusion, RPI at the femoral neck discriminated fracture cases from controls independent of BMD and traditional risk factors but dependent on location. The clinical RPI device may, therefore, supplement risk assessment, and requires testing in prospective cohorts and comparison between the clinically accessible tibia and the femoral neck. This article is protected by copyright. All rights reserved Sat, 01 Aug 2015 11:13:22 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2595 Oral bisphosphonates (BPs) are highly effective in preventing fractures and are recommended first-line therapies for patients with osteoporosis. We identified the incidence and predictors of oral BP treatment failure, defined as the incidence of ≥2 fractures while on treatment (≥2 FWOT) among users with high adherence. Fractures were considered after six months from treatment initiation and up to six months after discontinuation. Data from computerized records and pharmacy invoices were obtained from Sistema d‘Informació per al Desenvolupament de l‘Investigació en Atenció Primària (SIDIAP) (Catalonia, Spain) and Danish Health Registries (Denmark) for all incident users of oral BPs in 2006-2007 and 2000-2001 respectively. Fine and Gray survival models using backward-stepwise selection (p-entry 0.049; p-exit 0.10) and accounting for the competing risk of therapy cessation were used to identify predictors of ≥2 FWOT among patients having persisted with treatment ≥6months with overall medication possession ratio (MPR) ≥80%. Incidence of ≥2 FWOT was 2.4 (95% Confidence Interval (CI): 1.8-3.2) and 1.7 (95% CI: 1.2-2.2) per 1000 Person Years (PYs) within Catalonia and Denmark respectively. Older age was predictive of ≥2FWOT in both Catalonian and Danish cohorts: subhazard ratio (SHR) = 2.28 (95% CI: 1.11-4.68) and SHR = 2.61 (95% CI: 0.98-6.95) respectively for 65 to <80 years and SHR = 3.19 (95% CI: 1.33-7.69) and SHR = 4.88 (95% CI: 1.74-13.7) respectively for ≥80 years. Further significant predictors of ≥2 FWOT identified within only one cohort were dementia, SHR = 4.46 (95% CI: 1.02-19.4) (SIDIAP) and history of recent or older fracture, SHR = 3.40 (95% CI: 1.50-7.68) and SHR = 2.08 (95% CI: 1.04-4.15) respectively (Denmark). Even among highly adherent users of oral BP therapy, a minority sustain multiple fractures while on treatment. Older age was predictive of increased risk within both study populations, as was history of recent/old fracture and dementia within one but not both populations. Additional and/or alternative strategies should be investigated for these patients. This article is protected by copyright. All rights reserved Tue, 14 Jul 2015 11:04:49 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2520 Cathepsin K (CK), a lysosomal cysteine protease is highly expressed in mature osteoclasts and degrades type 1 collagen. Odanacatib (ODN) is a selective and reversible CK inhibitor that inhibits bone loss in preclinical and clinical studies. Although an anti-resorptive, ODN, does not suppress bone formation which led us to hypothesize that ODN may display restorative effect on the osteopenic bones. In a curative study, skeletally mature New Zealand rabbits were OVX and following induction of bone loss were given a steady-state exposure of ODN (9μM/day) for 14 weeks. Sham operated and OVX rabbits treated with alendronate (ALD), 17β-estradiol (E2) or PTH served as various controls. Efficacy was evaluated by assessing BMD, bone microarchitecture (using microcomputed tomography), fluorescent labeling of bone and biomechanical strength. Skeletal Ca/P ratio was measured by scanning electron microscopy (SEM) with X-ray microanalysis, crystallinity by X-ray diffraction, and bone mineral density distribution (tissue mineralization) by backscattered SEM. Between the sham and ODN-treated osteopenic groups, lumbar and femur metaphyseal BMD, Ca/P ratio, trabecular microstructure and geometric indices, vertebral compressive strength, trabecular lining cells, cortical parameters (femoral BMD, area and thickness, and periosteal deposition) and serum P1NP were largely comparable. Skeletal improvements in ALD or E2-treated groups fell significantly short of the sham/ODN/PTH group. However, the ODN group displayed reduced ductility and enhanced brittleness of central femur, which might have been contributed by higher crytallinity and tissue mineralization. Rabbit bone marrow stromal cells expressed CK and when treated with ODN displayed increased formation of mineralized nodules and decreased apoptosis in serum-deficient medium compared with control. In vivo, ODN did not suppress remodeling but inhibited osteoclast activity more than ALD. Taken together, we show that ODN reverses BMD, skeletal architecture and compressive strength in osteopenic rabbits however, increases crystallinity and tissue mineralization thus leading to increased cortical bone brittleness. This article is protected by copyright. All rights reserved Fri, 27 Mar 2015 16:57:47 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2704 As the most well-studied histone acetyltransferase (HAT) in yeast and mammals, general control nonderepressible 5 (GCN5) was documented to play essential roles in various developmental processes. However, little is known about its role in osteogenic differentiation of mesenchymal stem cells (MSCs). Here, we detected the critical function of GCN5 in osteogenic commitment of MSCs. In this role, the HAT activity of GCN5 was not required. Mechanistically, GCN5 repressed nuclear factor kappa B (NF-κB)-dependent transcription and inhibited the NF-κB signaling pathway. The impaired osteogenic differentiation by GCN5 knockdown was blocked by inhibition of NF-κB. Most importantly, the expression of GCN5 was decreased significantly in the bone tissue sections of ovariectomized mice or aged mice. Collectively, these results may point to the GCN5-NF-κB pathway as a novel potential molecular target for stem cell mediated regenerative medicine and the treatment of metabolic bone diseases such as osteoporosis. © 2015 American Society for Bone and Mineral Research. Wed, 30 Sep 2015 00:48:05 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2703 Although a higher protein intake, particularly from vegetable sources, has been shown to be associated with higher bone mineral density (BMD) the relative impact of specific amino acids on BMD and risk of osteoporosis remains to be determined. Mechanistic research suggests that a number of specific amino acids, including five nonessential amino acids—alanine, arginine, glutamic acid, glycine, and proline—may play a role in bone health, principally through improved production of insulin and insulin-like growth factor 1 and the synthesis of collagen and muscle protein. However to date, no previous studies have examined the associations between habitual intake of amino acids and direct measures of BMD and prevalence of osteoporosis or osteopenia, and no studies have examined this relationship in discordant identical twin-pairs. In these analyses of female monozygotic twin-pairs discordant for amino acid intake (n = 135), twins with higher intakes of alanine and glycine had significantly higher BMD at the spine than their co-twins with within-pair differences in spine-BMD of 0.012 g/cm2 (SE 0.01; p = 0.039) and 0.014 g/cm2 (SE 0.01; p = 0.026), respectively. Furthermore, in cross-sectional multivariable analyses of 3160 females aged 18 to 79 years, a higher intake of total protein was significantly associated with higher DXA-measured BMD at the spine (quartile Q4 to quartile Q1: 0.017 g/cm2, SE 0.01, p = 0.035) and forearm (Q4 to Q1: 0.010 g/cm2, SE 0.003, p = 0.002). Intake of six amino acids (alanine, arginine, glutamic acid, leucine, lysine, and proline) were associated with higher BMD at the spine and forearm with the strongest association observed for leucine (Q4 to Q1: 0.024 g/cm2, SE 0.01, p = 0.007). When intakes were stratified by protein source, vegetable or animal, prevalence of osteoporosis or osteopenia was 13% to 19% lower comparing extreme quartiles of vegetable intake for five amino acids (not glutamic acid or proline). These data provide evidence to suggest that intake of protein and several amino acids, including alanine and glycine, may be beneficial for bone health, independent of genetic background. © 2015 American Society for Bone and Mineral Research. Wed, 30 Sep 2015 00:45:20 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2707 Laparoscopic Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG) are common and effective methods to treat severe obesity, but these procedures can adversely influence bone metabolism and areal bone mineral density (aBMD). This was a prospective 24-month single-center interventional two-arm study in 220 women and similarly aged men (median age 40.7 years) with a body mass index (BMI) >38 kg/m2 after RYGB and SG procedures. Patients were randomized into: 1) an intervention group receiving: 28,000 IU cholecalciferol/wk for 8 weeks before bariatric surgery, 16,000 IU/wk and 1000 mg calciummonocitrate/d after surgery, daily BMI-adjusted protein supplementation and physical exercise (Nordic walking, strength perseverance, and equipment training); 2) a non-intervention group: no preoperative loading, nutritional supplementation, or obligatory physical exercise. At study endpoint, when comparing the intervention group to the non-intervention group, the relative percentage changes of serum levels of sclerostin (12.1% versus 63.8%), cross-linked C-telopeptide (CTX, 82.6% versus 158.3%), 25-OH vitamin D (13.4% versus 18.2%), phosphate (23.7% versus 32%, p < 0.001 for all), procollagen type 1 amino-terminal propeptide (P1NP, 12% versus 41.2%), intact parathyroid hormone (iPTH, –17.3% versus –7.6%), and Dickkopf-1 (–3.9% versus –8.9%, p < 0.05 for all) differed. The decline in lumbar spine, total hip and total body aBMD, changes in BMI, lean body mass (LBM), as well as changes in trabecular bone score (TBS) values (p < 0.005 for all) were less, but significantly, pronounced in the intervention group. We conclude that vitamin D loading and ongoing vitamin D, calcium, and BMI-adjusted protein supplementation in combination with physical exercise decelerates the loss of aBMD and LBM after bariatric surgery. Moreover, the well-known increases of bone turnover markers are less pronounced. © 2015 American Society for Bone and Mineral Research. Wed, 30 Sep 2015 00:44:23 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2701 Although caloric restriction (CR) could delay biologic aging in humans, it is unclear if this would occur at the cost of significant bone loss. We evaluated the effect of prolonged CR on bone metabolism and bone mineral density (BMD) in healthy younger adults. Two-hundred eighteen non-obese (body mass index [BMI] 25.1 ± 1.7 kg/m2), younger (age 37.9 ± 7.2 years) adults were randomly assigned to 25% CR (CR group, n = 143) or ad libitum (AL group, n = 75) for 2 years. Main outcomes were BMD and markers of bone turnover. Other outcomes included body composition, bone-active hormones, nutrient intake, and physical activity. Body weight (–7.5 ± 0.4 versus 0.1 ± 0.5 kg), fat mass (–5.3 ± 0.3 versus 0.4 ± 0.4 kg), and fat-free mass (–2.2 ± 0.2 versus –0.2 ± 0.2 kg) decreased in the CR group compared with AL (all between group p < 0.001). Compared with AL, the CR group had greater changes in BMD at 24 months: lumbar spine (–0.013 ± 0.003 versus 0.007 ± 0.004 g/cm2; p < 0.001), total hip (–0.017 ± 0.002 versus 0.001 ± 0.003 g/cm2; p < 0.001), and femoral neck (–0.015 ± 0.003 versus –0.005 ± 0.004 g/cm2; p = 0.03). Changes in bone markers were greater at 12 months for C-telopeptide (0.098 ± 0.012 versus 0.025 ± 0.015 μg/L; p < 0.001), tartrate-resistant acid phosphatase (0.4 ± 0.1 versus 0.2 ± 0.1 U/L; p = 0.004), and bone-specific alkaline phosphatase (BSAP) (–1.4 ± 0.4 versus –0.3 ± 0.5 U/L; p = 0.047) but not procollagen type 1 N-propeptide; at 24 months, only BSAP differed between groups (–1.5 ± 0.4 versus 0.9 ± 0.6 U/L; p = 0.001). The CR group had larger increases in 25-hydroxyvitamin D, cortisol, and adiponectin and decreases in leptin and insulin compared with AL. However, parathyroid hormone and IGF-1 levels did not differ between groups. The CR group also had lower levels of physical activity. Multiple regression analyses revealed that body composition, hormones, nutrients, and physical activity changes explained ∼31% of the variance in BMD and bone marker changes in the CR group. Therefore, bone loss at clinically important sites of osteoporotic fractures represents a potential limitation of prolonged CR for extending life span. Further long-term studies are needed to determine if CR-induced bone loss in healthy adults contributes to fracture risk and if bone loss can be prevented with exercise. © 2015 American Society for Bone and Mineral Research. Thu, 24 Sep 2015 02:35:55 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2690 The dental cementum covering the tooth root is similar to bone in several respects but remains poorly understood in terms of development and differentiation of cementoblasts, as well as the potential function(s) of cementocytes residing in the cellular cementum. It is not known if the cementocyte is a dynamic actor in cementum metabolism, comparable to the osteocyte in the bone. Cementocytes exhibit irregular spacing and lacunar shape, with fewer canalicular connections compared with osteocytes. Immunohistochemistry and quantitative PCR (qPCR) revealed that the in vivo expression profile of cementocytes paralleled that of osteocytes, including expression of dentin matrix protein 1 (Dmp1/DMP1), Sost/sclerostin, E11/gp38/podoplanin, Tnfrsf11b (osteoprotegerin [OPG]), and Tnfsf11 (receptor activator of NF-κB ligand [RANKL]). We used the Immortomouse+/−; Dmp1-GFP+/− mice to isolate cementocytes as Dmp1-expressing cells followed by immortalization using the interferon (IFN)-γ-inducible promoter driving expression of a thermolabile large T antigen to create the first immortalized line of cementocytes, IDG-CM6. This cell line reproduced the expression profile of cementocytes observed in vivo, including alkaline phosphatase activity and mineralization. IDG-CM6 cells expressed higher levels of Tnfrsf11b and lower levels of Tnfsf11 compared with IDG-SW3 osteocytes, and under fluid flow shear stress, IDG-CM6 cells significantly increased OPG while decreasing RANKL, leading to a significantly increased OPG/RANKL ratio, which would inhibit osteoclast activation. These studies indicate similarities yet potentially important differences in the function of cementocytes compared with osteocytes and support cementocytes as mechanically responsive cells. © 2015 American Society for Bone and Mineral Research. Wed, 23 Sep 2015 04:50:12 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2696 Bone strength depends on the amount of bone, typically expressed as bone mineral density (BMD), determined by dual-energy X-ray absorptiometry (DXA), and on bone quality. Bone quality is a multifactorial entity including bone structural and material compositional properties. The purpose of the present study was to examine whether bone material composition properties at actively-forming trabecular bone surfaces in health are dependent on subject age, and to contrast them with postmenopausal osteoporosis patients. To achieve this, we analyzed by Raman microspectroscopy iliac crest biopsy samples from healthy subjects aged 1.5 to 45.7 years, paired biopsy samples from females before and immediately after menopause aged 46.7 to 53.6 years, and biopsy samples from placebo-treated postmenopausal osteoporotic patients aged 66 to 84 years. The monitored parameters were as follows: the mineral/matrix ratio; the mineral maturity/crystallinity (MMC); nanoporosity; the glycosaminoglycan (GAG) content; the lipid content; and the pyridinoline (Pyd) content. The results indicate that these bone quality parameters in healthy, actively-forming trabecular bone surfaces are dependent on subject age at constant tissue age, suggesting that with advancing age the kinetics of maturation (either accumulation, or posttranslational modifications, or both) change. For most parameters, the extrapolation of models fitted to the individual age dependence of bone in healthy individuals was in rough agreement with their values in postmenopausal osteoporotic patients, except for MMC, lipid, and Pyd content. Among these three, Pyd content showed the greatest deviation between healthy aging and disease, highlighting its potential to be used as a discriminating factor. © 2015 American Society for Bone and Mineral Research. Wed, 23 Sep 2015 04:48:54 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2695 Wed, 23 Sep 2015 04:47:41 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2697 Somewhat paradoxically, fracture risk, which depends on applied loads and bone strength, is elevated in both anorexia nervosa and obesity at certain skeletal sites. Factor-of-risk (Φ), the ratio of applied load to bone strength, is a biomechanically based method to estimate fracture risk; theoretically, higher Φ reflects increased fracture risk. We estimated vertebral strength (linear combination of integral volumetric bone mineral density [Int.vBMD] and cross-sectional area from quantitative computed tomography [QCT]), vertebral compressive loads, and Φ at L4 in 176 women (65 anorexia nervosa, 45 lean controls, and 66 obese). Using biomechanical models, applied loads were estimated for: 1) standing; 2) arms flexed 90°, holding 5 kg in each hand (holding); 3) 45° trunk flexion, 5 kg in each hand (lifting); 4) 20° trunk right lateral bend, 10 kg in right hand (bending). We also investigated associations of Int.vBMD and vertebral strength with lean mass (from dual-energy X-ray absorptiometry [DXA]) and visceral adipose tissue (VAT, from QCT). Women with anorexia nervosa had lower, whereas obese women had similar, Int.vBMD and estimated vertebral strength compared with controls. Vertebral loads were highest in obesity and lowest in anorexia nervosa for standing, holding, and lifting (p < 0.0001) but were highest in anorexia nervosa for bending (p < 0.02). Obese women had highest Φ for standing and lifting, whereas women with anorexia nervosa had highest Φ for bending (p < 0.0001). Obese and anorexia nervosa subjects had higher Φ for holding than controls (p < 0.03). Int.vBMD and estimated vertebral strength were associated positively with lean mass (R = 0.28 to 0.45, p ≤ 0.0001) in all groups combined and negatively with VAT (R = –[0.36 to 0.38], p < 0.003) within the obese group. Therefore, women with anorexia nervosa had higher estimated vertebral fracture risk (Φ) for holding and bending because of inferior vertebral strength. Despite similar vertebral strength as controls, obese women had higher vertebral fracture risk for standing, holding, and lifting because of higher applied loads from higher body weight. Examining the load-to-strength ratio helps explain increased fracture risk in both low-weight and obese women. © 2015 American Society for Bone and Mineral Research. Thu, 17 Sep 2015 06:50:41 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2700 Wed, 16 Sep 2015 02:16:38 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2699 Wed, 16 Sep 2015 02:15:09 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2694 Mon, 14 Sep 2015 01:08:32 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2691 Although fibroblast growth factor (FGF) 23 was recently identified as a phosphatonin that influences vitamin D metabolism, the underlying signaling mechanisms remain unclear. FGF23 elevates the renal levels of membrane-associated klotho as well as soluble klotho. Klotho is expressed on distal tubules. Upon enzymatic cleavage, soluble klotho is released into the renal interstitial space and then into the systemic circulation. The expression of 25-hydroxyvitamin D3 1α-hydroxylase (1-OH) on proximal tubular cells is controlled by parathyroid hormone (PTH). Klotho binds to various membrane proteins to alter their function. Here, the interaction between the PTH receptor and klotho was studied using various approaches, including immunoprecipitation, in vitro cell culture, and in vivo animal experiments. Immunoprecipitation studies demonstrate, for the first time, that recombinant human klotho protein interacts with human PTH receptors to inhibit the binding of human PTH. Furthermore, when applied to human proximal tubular cells, recombinant human klotho suppresses PTH-stimulated generation of inositol trisphosphate in vitro. Moreover, PTH-induced increase of cyclic AMP secretion and 1α,25-dihydroxyvitamin D3 (1,25VD) was attenuated by recombinant human klotho in vivo. In addition, recombinant human klotho inhibits the expression of 1-OH by PTH both in vitro and in vivo. These results suggest that free klotho mediates the FGF23-induced inhibition of 1,25VD synthesis. © 2015 American Society for Bone and Mineral Research. Fri, 11 Sep 2015 02:20:53 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2687 Protein phosphorylation regulates a wide variety of cellular processes. Thus, we hypothesize that single-nucleotide polymorphisms (SNPs) that may modulate protein phosphorylation could affect osteoporosis risk. Based on a previous conventional genome-wide association (GWA) study, we conducted a three-stage meta-analysis targeting phosphorylation-related SNPs (phosSNPs) for femoral neck (FN)-bone mineral density (BMD), total hip (HIP)-BMD, and lumbar spine (LS)-BMD phenotypes. In stage 1, 9593 phosSNPs were meta-analyzed in 11,140 individuals of various ancestries. Genome-wide significance (GWS) and suggestive significance were defined by α = 5.21 × 10–6 (0.05/9593) and 1.00 × 10–4, respectively. In stage 2, nine stage 1–discovered phosSNPs (based on α = 1.00 × 10–4) were in silico meta-analyzed in Dutch, Korean, and Australian cohorts. In stage 3, four phosSNPs that replicated in stage 2 (based on α = 5.56 × 10–3, 0.05/9) were de novo genotyped in two independent cohorts. IDUA rs3755955 and rs6831280, and WNT16 rs2707466 were associated with BMD phenotypes in each respective stage, and in three stages combined, achieving GWS for both FN-BMD (p = 8.36 × 10–10, p = 5.26 × 10–10, and p = 3.01 × 10–10, respectively) and HIP-BMD (p = 3.26 × 10–6, p = 1.97 × 10–6, and p = 1.63 × 10–12, respectively). Although in vitro studies demonstrated no differences in expressions of wild-type and mutant forms of IDUA and WNT16B proteins, in silico analyses predicts that WNT16 rs2707466 directly abolishes a phosphorylation site, which could cause a deleterious effect on WNT16 protein, and that IDUA phosSNPs rs3755955 and rs6831280 could exert indirect effects on nearby phosphorylation sites. Further studies will be required to determine the detailed and specific molecular effects of these BMD-associated non-synonymous variants. © 2015 American Society for Bone and Mineral Research. Fri, 11 Sep 2015 02:20:36 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2692 Age-related loss of skeletal muscle mass and strength are risk factors for sarcopenia, osteoporosis, falls, fractures, frailty, and mortality. Dietary magnesium (Mg) could play a role in prevention of age-related loss of skeletal muscle mass, power, and strength directly through physiological mechanisms or indirectly through an impact on chronic low-grade inflammation, itself a risk factor for loss of skeletal muscle mass and strength. In a cross-sectional study of 2570 women aged 18 to 79 years, we examined associations between intakes of Mg, estimated using a food-frequency questionnaire (FFQ), dual-energy X-ray absorptiometry (DXA)-derived measures of muscle mass (fat-free mass as a percentage of body weight [FFM%], fat-free mass index [FFMI, kg/m2]), leg explosive power (LEP), and grip strength (n = 949 only). We also examined associations between circulating hs-CRP (C-reactive protein) and muscle mass and LEP, and explored the potential attenuation of these relationships by Mg. We compared our findings with those of age and protein intake. Endpoints were calculated by quintile of Mg and adjusted for relevant confounders. Significant positive associations were found between a higher Mg and indices of skeletal muscle mass and LEP, and also with hs-CRP, after adjustment for covariates. Contrasting extreme quintiles of Mg intake showed differences of 2.6% for FFM% (p trend < 0.001), 0.4 kg/m2 for FFMI (p trend = 0.005), and 19.6 watts/kg for LEP (p trend < 0.001). Compared with protein, these positive associations were 7 times greater for FFM% and 2.5 times greater for LEP. We also found that higher hs-CRP was negatively associated with skeletal muscle mass and, in statistical modeling, that a higher dietary Mg attenuated this negative relationship by 6.5%, with greater attenuation in women older than 50 years. No association was found between Mg and grip strength. Our results suggest that dietary magnesium may aid conservation of age-related loss of skeletal muscle mass and power in women of all ages. © 2015 American Society for Bone and Mineral Research. Fri, 11 Sep 2015 02:18:54 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2613 Head injuries resulting from motor vehicle crashes (MVC) are extremely common, yet the details of the mechanism of injury remain to be well characterized. Skull deformation is believed to be a contributing factor to some types of traumatic brain injury (TBI). Understanding biomechanical contributors to skull deformation would provide further insight into the mechanism of head injury resulting from blunt trauma. In particular, skull thickness is thought be a very important factor governing deformation of the skull and its propensity for fracture. Previously, age- and sex-based skull cortical thickness changes were difficult to evaluate based on the need for cadaveric skulls. In this cross-sectional study, skull thickness changes with age and sex have been evaluated at homologous locations using a validated cortical density-based algorithm to accurately quantify cortical thickness from 123 high-resolution clinical computed tomography (CT) scans. The flat bones of the skull have a sandwich structure; therefore, skull thickness was evaluated for the inner and outer tables as well the full thickness. General trends indicated an increase in the full skull thickness, mostly attributed to an increase in the thickness of the diploic layer; however, these trends were not found to be statistically significant. There was a significant relationship between cortical thinning and age for both tables of the frontal, occipital, and parietal bones ranging between a 36% and 60% decrease from ages 20 to 100 years in females, whereas males exhibited no significant changes. Understanding how cortical and full skull thickness changes with age from a wide range of subjects can have implications in improving the biofidelity of age- and sex-specific finite element models and therefore aid in the prediction and understanding of TBI from impact and blast injuries. © 2015 American Society for Bone and Mineral Research. Tue, 08 Sep 2015 03:02:06 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2693 Osteonecrosis of the jaw (ONJ) is a serious adverse drug event that was initially reported with intravenous bisphosphonates (BPs) and more recently with other classes of drugs such as receptor activator of NF-κB ligand (RANKL) inhibitor, antiangiogenic agents, and mammalian target of rapamycin (m-TOR) inhibitors. The purpose of this study is to analyze the ONJ cases and the associated drugs in the US Food and Drug Administration's adverse event reporting system (FAERS). The FAERS database was queried for the adverse drug events reported from the first quarter of 2010 to the first quarter of 2014. The reporting odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for each queried drug. A total of 17,119 unique ONJ cases were identified. In the overall analysis, the drugs with the highest reporting ORs were BPs: pamidronate (OR = 498.9), zoledronate (OR = 171.7), and alendronate (OR = 63.6), whereas denosumab had lower ORs than all the BPs except for etidronate. The antiangiogenic and m-TOR inhibitors had the lowest ORs. In cancer patients who were treated for prevention of skeletal-related events (SREs), the reporting ORs for zoledronate and denosumab were 125.2 and 4.9, respectively. In patients with osteoporosis, the ORs were 1.1 (1.0–1.18) for zoledronate and 0.63 (0.56–0.70) for denosumab, respectively. Our analysis of the FAERS database showed that the intravenous BPs were associated with the highest risk for ONJ, RANKL inhibitor was associated with risk comparable to BPs used for osteoporosis such as etidronate, and the antiangiogenic agents and m-TOR inhibitors were associated with the lowest risk for ONJ. The high risk for ONJ with zoledronate and denosumab was mainly observed in those who were treated for prevention of SREs, whereas there was limited evidence for such risk in those who were treated for osteoporosis. © 2015 American Society for Bone and Mineral Research. Fri, 04 Sep 2015 01:27:42 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2607 Low-energy fractures are frequent complications in type 1 diabetes mellitus patients (T1DM). Modifications of bone intrinsic composition might be a potential cause of fragility observed in diabetic subjects. Advanced glycation end products (AGEs) were found in numerous connective tissues from T1DM patients. However, whether AGEs are present at high levels in bone matrix from diabetic subjects is unknown. Moreover, whether elevated AGEs in the bone matrix impair mineralization has not been addressed in humans. The purposes of this study were 1) to determine whether bone matrix from fracturing and nonfracturing T1DM contained more AGEs than bone from healthy patients (CTL), and 2) to compare the degree of mineralization of bone and hardness between fracturing and nonfracturing T1DM versus CTL. We analyzed iliac crest bone biopsies from 5 fracturing T1DM patients, 5 nonfracturing T1DM patients, and 5 healthy subjects, all age- and sex-matched. AGEs (pentosidine) in bone matrix was measured by high-performance liquid chromatography separately in trabecular and cortical bone. The degree of mineralization of bone (DMB) was assessed by digitized microradiography, and mechanical properties by micro- and nanohardness tests. Trabecular bone from fracturing T1DM exhibited significantly higher levels of pentosidine than CTL (p = 0.04) and was more mineralized than nonfracturing T1DM (p = 0.04) and CTL (p = 0.04). Trabecular bone was not significantly different in pentosidine between nonfracturing T1DM and CTL. Cortical bone from nonfracturing T1DM was not significantly different from CTL. Positive correlations were found between HbA1c and pentosidine (r' = 0.79, p < 0.003) and between HbA1c and DMB (r' = 0.64, p < 0.02). Both modifications could lead to less flexible bone (reduced modulus of elasticity) and a tendency toward low-energy fractures in T1DM patients. © 2015 American Society for Bone and Mineral Research. Fri, 04 Sep 2015 01:27:23 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2597 Red blood cell distribution width (RDW) has been found to be a significant prognostic factor of mortality in many cardiovascular diseases. However, a link between RDW at admission with long-term mortality in the hip fracture population has not been well established. Therefore, we sought to evaluate the long-term prognostic value of RDW in a well-defined hip fracture cohort, and to compare the effect of RDW in patients with and without anemia. A prospective cohort study was performed on 1479 hip fracture patients admitted at the General Hospital of Chinese PLA between January 2000 and October 2011 with a follow-up study over a 2-year period. A total of 1479 patients were used for the evaluation of 2-year all-cause mortality, while 804 patients with more than 4 years of follow-up were extracted for further evaluation of 4-year all-cause mortality. Cox proportional regression was used to evaluate the association between admission RDW and long-term mortality, adjusting for potential confounding variables. Higher RDW values were strongly associated with increased all-cause mortality. After adjusting for age, mean corpuscular volume, admission hemoglobin, comorbidities, and complications, RDW had a significant independent association with both 2-year mortality with a hazard ratio (HR) of 1.183 (95% confidence interval [CI], 1.017 to 1.376) and 4-year mortality with an HR of 1.244 (95% CI, 1.052 to 1.471). In stratified analysis, the effect of RDW was even more pronounced, with 2-year mortality HR of 1.341 (95% CI, 1.095 to 1.643) and 4-year mortality HR of 1.345 (95% CI, 1.071 to 1.688) in non-anemic patients. In non-anemic patients, elevated RDW values are significantly associated with increased odds of all-cause mortality, implying that RDW may be a possible laboratory biomarker for risk stratification in non-anemic hip fracture patients. Further studies are needed to confirm the current finding in different and larger hip fracture cohorts. © 2015 American Society for Bone and Mineral Research. Thu, 03 Sep 2015 01:27:13 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2610 Trabecular bone score (TBS) rests on the textural analysis of dual-energy X-ray absorptiometry (DXA) to reflect the decay in trabecular structure characterizing osteoporosis. Yet, its discriminative power in fracture studies remains incomprehensible because prior biomechanical tests found no correlation with vertebral strength. To verify this result possibly owing to an unrealistic setup and to cover a wide range of loading scenarios, the data from three previous biomechanical studies using different experimental settings were used. They involved the compressive failure of 62 human lumbar vertebrae loaded 1) via intervertebral discs to mimic the in vivo situation (“full vertebra”); 2) via the classical endplate embedding (“vertebral body”); or 3) via a ball joint to induce anterior wedge failure (“vertebral section”). High-resolution peripheral quantitative computed tomography (HR-pQCT) scans acquired from prior testing were used to simulate anterior-posterior DXA from which areal bone mineral density (aBMD) and the initial slope of the variogram (ISV), the early definition of TBS, were evaluated. Finally, the relation of aBMD and ISV with failure load (Fexp) and apparent failure stress (σexp) was assessed, and their relative contribution to a multilinear model was quantified via ANOVA. We found that, unlike aBMD, ISV did not significantly correlate with Fexp and σexp, except for the “vertebral body” case (r2 = 0.396, p = 0.028). Aside from the “vertebra section” setup where it explained only 6.4% of σexp (p = 0.037), it brought no significant improvement to aBMD. These results indicate that ISV, a replica of TBS, is a poor surrogate for vertebral strength no matter the testing setup, which supports the prior observations and raises a fortiori the question of the deterministic factors underlying the statistical relationship between TBS and vertebral fracture risk. © 2015 American Society for Bone and Mineral Research. Thu, 03 Sep 2015 01:26:51 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2608 There is increasing evidence that physical exercise can prevent fractures in postmenopausal women. However, even with careful adaptation of the exercise program to subjects' changing bone, health, and fitness status, effectivity may still decrease over the time. This could be specifically the case where the limitations of higher age collide with the specification of the exercise program. Thus, the aim of this study was to monitor bone mineral density (BMD) changes over a 16-year period of supervised exercise. Our hypothesis was that BMD differences at lumbar spine (LS) and femoral neck (FN) between exercisers (EG) and nontraining controls (CG) increased throughout the intervention with significant differences for each of the four 4-year observation periods. Sixty-seven (EG, n = 39 versus CG, n = 28) initially early-postmenopausal osteopenic women of the Erlangen Fitness and Osteoporosis Prevention Study (EFOPS) with complete BMD data sets for baseline (1998) and 4-, 8-, 12-, and 16-year follow-up were included in the analysis. The exercise protocol initially focused on a high-intensity strategy that addressed bone but increasingly shifted to a more comprehensive intervention. LS-BMD differences between the EG and CG continuously increased (year 4: 2.4% (95%- Confidence Interval 1.0% to 3.8%), year 8: 3.1% (1.6% to 4.7%), year 12: 3.9% (1.9% to 5.8), year 16: 4.5% (2.5% to 6.6%). Correspondingly, rising differences for FN-BMD (0.9% (–0.2% to 2.1%) versus 1.9% (0.4% to 3.3%) versus 2.0% (0.5% to 3.8%) versus 3.0% (1.0% to 5.0%)) were observed. However, in contrast to our hypothesis, group differences within the four 4-year periods were not consistently significant (LS, p = 0.001 to 0.097; FN, p = 0.026 to 0.673); further, BMD kinetics among the groups varied between LS and FN. Of particular importance, significant differences (p ≤ 0.030) for both regions were still found in the final period. We conclude that exercise—even when adapted for subjects' decreasing bone, health, and fitness status—is consistently effective in favorably affecting BMD in (initially) early-postmenopausal osteopenic women without any leveling-off effect after 16 years of exercise. © 2015 American Society for Bone and Mineral Research. Thu, 03 Sep 2015 01:26:29 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2609 In hypoparathyroidism, areal bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA) is above average, and skeletal indices by bone biopsy are abnormal. We used high-resolution peripheral quantitative computed tomography (HRpQCT) and finite element analyses (FEA) to further investigate skeletal microstructure and estimated bone strength. We studied 60 hypoparathyroid subjects on conventional therapy using DXA, HRpQCT, and FEA of the distal radius and tibia compared with normative controls from the Canadian Multicentre Osteoporosis Study. In hypoparathyroid women and men, areal BMD was above average at the lumbar spine and hip sites by DXA; radial BMD was also above average in hypoparathyroid women. Using HRpQCT, cortical volumetric BMD was increased in the hypoparathyroid cohort compared with controls at both the radius and tibia. Cortical porosity was reduced at both sites in pre- and postmenopausal women and at the tibia in young men with a downward trend at the radius in men. At the tibia, trabecular number was increased in premenopausal women and men and trabecular thickness was lower in women. Ultimate stress and failure load at both sites for the hypoparathyroid subjects were similar to controls. Using a linear regression model, at both radius and tibia, each increment in age decreased ultimate stress and failure load, whereas each increment in duration of hypoparathyroidism increased these same indices. These results provide additional evidence for the critical role of parathyroid hormone in regulating skeletal microstructure. Longer disease duration may mitigate the adverse effects of age on estimated bone strength in hypoparathyroidism. © 2015 American Society for Bone and Mineral Research. Thu, 03 Sep 2015 01:26:08 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2689 Advanced aging is associated with increased risk of bone fracture, especially within the vertebrae, which exhibit significant reductions in trabecular bone structure. Aging is also associated with a reduction in circulating levels of insulin-like growth factor (IGF-1). Studies have suggested that the reduction in IGF-1 compromises healthspan, whereas others report that loss of IGF-1 is beneficial because it increases healthspan and lifespan. To date, the effect of decreases in circulating IGF-1 on vertebral bone aging has not been thoroughly investigated. Here, we delineate the consequences of a loss of circulating IGF-1 on vertebral bone aging in male and female Igff/f mice. IGF-1 was reduced at multiple specific time points during the mouse lifespan: early in postnatal development (crossing albumin–cyclic recombinase [Cre] mice with Igff/f mice); and in early adulthood and in late adulthood using hepatic-specific viral vectors (AAV8-TBG-Cre). Vertebrae bone structure was analyzed at 27 months of age using micro–computed tomography (μCT) and quantitative bone histomorphometry. Consistent with previous studies, both male and female mice exhibited age-related reductions in vertebral bone structure. In male mice, reduction of circulating IGF-1 induced at any age did not diminish vertebral bone loss. Interestingly, early-life loss of IGF-1 in females resulted in a 67% increase in vertebral bone volume fraction, as well as increased connectivity density and increased trabecular number. The maintenance of bone structure in the early-life IGF-1–deficient females was associated with increased osteoblast surface and an increased ratio of osteoprotegerin/receptor-activator of NF-κB-ligand (RANKL) levels in circulation. Within 3 months of a loss of IGF-1, there was a 2.2-fold increase in insulin receptor expression within the vertebral bones of our female mice, suggesting that local signaling may compensate for the loss of circulating IGF-1. Together, these data suggest the age-related loss of vertebral bone density in females can be reduced by modifying circulating IGF-1 levels early in life. © 2015 American Society for Bone and Mineral Research. Thu, 03 Sep 2015 01:25:46 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2611 Fracture risk estimates are usually based on femoral neck (FN) BMD. It is unclear how to address T-score discordance, where lumbar spine (LS) T-score is lower than FN T-score. The objective of this work was to examine the impact of LS BMD on fracture risk, in individuals with lower LS T-score than FN T-score. Participants aged 60+ years from the Dubbo Osteoporosis Epidemiology Study with LS and FN BMD measured at first visit, and were followed from 1989 to 2014. Five-hundred and seventy-three (573) of 2270 women and 131 of 1373 men had lower LS than FN T-score by ≥0.6 standard deviation (SD) (low-LS group based on least significant change). In low-LS women, each 1 SD lower LS T-score than FN was associated with a 30% increase in fracture risk (hazard ratio [HR] 1.30; 95% CI, 1.11 to 1.45). For low-LS men there was a 20% nonsignificant increase in fracture risk for each 1 SD lower LS than FN T-score (HR 1.20; 95% CI, 0.10 to 1.67). Low-LS women had greater absolute fracture risks than the rest of the women. This increased risk was more apparent for lower levels of FN T-score and in older age groups. At an FN T-score of –2, low-LS women had a 3%, 10%, and 23% higher 5-year absolute fracture risk than non-low LS women in the 60 to 69 year, 70 to 79 year, and 80+ years age-groups, respectively. Furthermore, an osteoporotic LS T-score increased 5-year absolute fracture risk for women with normal or osteopenic FN T-score by 10% to 13%. Men in the low-LS group had very few fractures; therefore, a meaningful analyses of fracture risk could not be conducted. This study shows the significant contribution of lower LS BMD to fracture risk over and above FN BMD in women. A LS BMD lower than FN BMD should be incorporated into fracture risk calculators at least for women in older age-groups. © 2015 American Society for Bone and Mineral Research. Thu, 03 Sep 2015 01:25:18 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2688 This 4-year cluster randomized controlled trial of 365 boys and 362 girls (mean age 8.1 ± 0.3 years) from grade 2 in 29 primary schools investigated the effects of a specialist-taught physical education (PE) program on bone strength and body composition. All children received 150 min/week of common practice (CP) PE from general classroom teachers but in 13 schools 100 min/week of CP PE was replaced by specialized-led PE (SPE) by teachers who emphasized more vigorous exercise/games combined with static and dynamic postural activities involving muscle strength. Outcome measures assessed in grades 2, 4, and 6 included: total body bone mineral content (BMC), lean mass (LM), and fat mass (FM) by DXA, and radius and tibia (4% and 66% sites) bone structure, volumetric density and strength, and muscle cross-sectional area (CSA) by pQCT. After 4-years, gains in total body BMC, FM, and muscle CSA were similar between the groups in both sexes, but girls in the SPE group experienced a greater gain in total body LM (mean 1.0 kg; 95% CI, 0.2 to 1.9 kg). Compared to CP, girls in the SPE group also had greater gains in cortical area (CoA) and cortical thickness (CoTh) at the mid-tibia (CoA, 5.0% [95% CI, 0.2% to 1.9%]; CoTh, 7.5% [95% CI, 2.4% to 12.6%]) and mid-radius (CoA, 9.3% [95% CI, 3.5% to 15.1%]; CoTh, 14.4% [95% CI, 6.1% to 22.7%]), whereas SPE boys had a 5.2% (95% CI, 0.4% to 10.0%) greater gain in mid-tibia CoTh. These benefits were due to reduced endocortical expansion. There were no significant benefits of SPE on total bone area, cortical density or bone strength at the mid-shaft sites, nor any appreciable effects at the distal skeletal sites. This study indicates that a specialist-led school-based PE program improves cortical bone structure, due to reduced endocortical expansion. This finding challenges the notion that periosteal apposition is the predominant response of bone to loading during the prepubertal and early-pubertal period. © 2015 American Society for Bone and Mineral Research. Thu, 03 Sep 2015 01:24:31 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2606 Fibroblast growth factor-23 (Fgf23) is a bone-derived hormone, suppressing phosphate reabsorption and vitamin D hormone (1,25(OH)2D3) production in the kidney. It has long been an enigma why lack of Fgf23 or of Klotho, the coreceptor for Fgf23, leads to severe impairment in bone mineralization despite the presence of hypercalcemia and hyperphosphatemia. Using Fgf23-/- or Klotho-/- mice together with compound mutant mice lacking both Fgf23 or Klotho and a functioning vitamin D receptor, we show that in Klotho-/- mice the mineralization defect is solely driven by 1,25(OH)2D3-induced upregulation of the mineralization-inhibiting molecules osteopontin and pyrophosphate in bone. In Fgf23-/- mice, the mineralization defect has two components, a 1,25(OH)2D3-driven component similar to Klotho-/- mice and a component driven by lack of Fgf23, causing additional accumulation of osteopontin. We found that FGF23 regulates osteopontin secretion indirectly by suppressing alkaline phosphatase transcription and phosphate production in osteoblastic cells, acting through FGF receptor-3 in a Klotho-independent manner. Hence, FGF23 secreted from osteocytes may form an autocrine/paracrine feedback loop for the local fine-tuning of bone mineralization. © 2015 American Society for Bone and Mineral Research. Sat, 29 Aug 2015 07:46:56 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2612 The small molecule WHI-131 is a potent therapeutic agent with anti-inflammatory, antiallergic, and antileukemic potential. However, the regulatory effects of WHI-131 on osteoblast and osteoclast activity are unclear. We examined the effects of WHI-131 on osteoblast and osteoclast differentiation with respect to bone remodeling. The production of receptor activator of nuclear factor kappa-B ligand (RANKL) by osteoblasts in response to interleukin (IL)-1 or IL-6 stimulation decreased by 56.8% or 50.58%, respectively, in the presence of WHI-131. WHI-131 also abrogated the formation of mature osteoclasts induced by IL-1 or IL-6 stimulation. Moreover, WHI-131 treatment decreased RANKL-induced osteoclast differentiation of bone marrow-derived macrophages, and reduced the resorbing activity of mature osteoclasts. WHI-131 further decreased the mRNA and protein expression levels of c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) by almost twofold, and significantly downregulated the mRNA expression of the following genes: tartrate-resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR), DC-STAMP, OC-STAMP, ATP6v0d2, and cathepsin K (CtsK) compared with the control group. WHI-131 further suppressed the phosphorylation of protein kinase B (Akt) and degradation of inhibitor of kappa B (IκB); Ca2+ oscillation was also affected, and phosphorylation of the C-terminal Src kinase (c-Src)–Bruton agammaglobulinemia tyrosine kinase (Btk)–phospholipase C gamma 2 (PLCγ2) (c-Src-Btk-PLCg2 calcium signaling pathway) was inhibited following WHI-131 treatment. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway was activated by WHI-131, accompanied by phosphorylation of STAT3 Ser727 and dephosphorylation of STAT6. In osteoblasts, WHI-131 caused an approximately fourfold increase in alkaline phosphatase activity and Alizarin Red staining intensity. Treatment with WHI-131 increased the mRNA expression levels of genes related to osteoblast differentiation, and induced the phosphorylation of Akt, p38, and Smad1/5/8. Furthermore, 5-week-old ICR mice treated with WHI-131 exhibited antiresorbing effects in a lipopolysaccharide-induced calvaria bone loss model in vivo and increased bone-forming activity in a calvarial bone formation model. Therefore, the results of this study show that WHI-131 plays a dual role by inhibiting osteoclast differentiation and promoting osteoblast differentiation. Thus, WHI-131 could be a useful pharmacological agent to treat osteoporosis by promoting bone growth and inhibiting resorption. © 2015 American Society for Bone and Mineral Research. Sat, 29 Aug 2015 07:45:57 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2604 We conducted a genome-wide association study of low bone mineral density (BMD) at the hip and spine utilizing sequence variants found through whole-genome sequencing of 2636 Icelanders. We found two rare missense mutations, p.Gly496Ala and p.Gly703Ser, in the COL1A2 gene that associate with measures of osteoporosis in Icelanders. Mutations in COL1A2 are known to cause the autosomal dominant disorder osteogenesis imperfecta. Both variants associate with low BMD and with osteoporotic fractures. p.Gly496Ala (frequency of 0.105%) shows the strongest association with low BMD at the spine (p = 1.8 × 10−7, odds ratio [OR] = 4.61 [95% confidence interval (CI) 2.59, 8.18]), whereas p.Gly703Ser (frequency of 0.050%) is most strongly associated with low BMD at the hip (p = 1.9 × 10−8, OR = 9.34 [95% CI 4.28, 20.3]). Association with fractures was p = 2.2 × 10−5, OR = 3.75 (95% CI 2.03, 6.93) and p = 0.0023, OR = 4.32 (95% CI 1.69, 11.1), respectively. The carriers of these variants do not have signs of osteogenesis imperfecta other than low BMD, demonstrating that similar mutations in COL1A2 can affect skeletal phenotypes in more than one way. © 2015 American Society for Bone and Mineral Research. Sat, 29 Aug 2015 07:44:35 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2596 Metabolic and bone effects were investigated in growing (G, n = 45) and mature (M, n = 45) rats fed a high-fat/high-sucrose diet (HFS) isocaloric to the chow diet of controls (C, n = 30 per group). At week 19, a subset of 15 rats in each group (HFS or C, at both ages) was analyzed. Then one-half of the remaining 30 HFS rats in each groups continued HFS and one-half were shifted to C until week 27. Although no serum or bone marrow inflammation was seen, HFS increased visceral fat, serum leptin and insulin at week 19 and induced further alterations in lipid profile, serum adiponectin, and TGFβ1, TIMP1, MMP2, and MMP9, suggesting a prediabetic phenotype and cardiovascular dysfunction at week 27 more pronounced in M than G. These events were associated with dramatic reduction of osteoclastic and osteoid surfaces with accelerated mineralizing surfaces in both HFS age groups. Mineral metabolism and its major regulators were disturbed, leading to hyperphosphatemia and hypocalcemia. These changes were associated with bone alterations in the weight-bearing tibia, not in the non-weight-bearing vertebra. Indeed in fat rats, tibia trabecular bone accrual increased in G whereas loss of trabecular bone in M was alleviated. At diaphysis cortical porosity increased in G and even more in M at week 27. After the diet switch, metabolic and bone cellular disturbances fully reversed in G, but not in M. Trabecular benefit of the obese was preserved in both age groups and in M the age-related bone loss was even lighter after the diet switch than in prolonged HFS. At the diaphysis, cortical porosity normalized in G but not in M. Hypocalcemia in G and M was irreversible. Thus, the mild metabolic syndrome induced by isocaloric HFS is able to alter bone cellular activities and mineral metabolism, reinforce trabecular bone, and affect cortical bone porosity in an irreversible manner in older rats. © 2015 American Society for Bone and Mineral Research. Fri, 28 Aug 2015 02:34:17 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2602 Bone loss and increased marrow adiposity are hallmarks of aging skeletons. Conditional deletion of histone deacetylase 3 (Hdac3) in murine osteochondroprogenitor cells causes osteopenia and increases marrow adiposity, even in young animals, but the origins of the increased adiposity are unclear. To explore this, bone marrow stromal cells (BMSCs) from Hdac3-depleted and control mice were cultured in osteogenic medium. Hdac3-deficient cultures accumulated lipid droplets in greater abundance than control cultures and expressed high levels of genes related to lipid storage (Fsp27/Cidec, Plin1) and glucocorticoid metabolism (Hsd11b1) despite normal levels of Pparγ2. Approximately 5% of the lipid containing cells in the wild-type cultures expressed the master osteoblast transcription factor Runx2, but this population was threefold greater in the Hdac3-depleted cultures. Adenoviral expression of Hdac3 restored normal gene expression, indicating that Hdac3 controls glucocorticoid activation and lipid storage within osteoblast lineage cells. HDAC3 expression was reduced in bone cells from postmenopausal as compared to young women, and in osteoblasts from aged as compared to younger mice. Moreover, phosphorylation of S424 in Hdac3, a posttranslational mark necessary for deacetylase activity, was suppressed in osseous cells from old mice. Thus, concurrent declines in transcription and phosphorylation combine to suppress Hdac3 activity in aging bone, and reduced Hdac3 activity in osteochondroprogenitor cells contributes to increased marrow adiposity associated with aging. © 2015 American Society for Bone and Mineral Research. Thu, 20 Aug 2015 06:44:47 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2600 1,25 Dihydroxyvitamin D3 (1,25(OH)2D) increases intestinal Ca absorption when dietary Ca intake is low by inducing gene expression through the vitamin D receptor (VDR). 1,25(OH)2D-regulated Ca absorption has been studied extensively in the small intestine, but VDR is also present in the large intestine. Our goal was to determine the impact of large intestinal VDR deletion on Ca and bone metabolism. We used transgenic mice expressing Cre-recombinase driven by the 9.5-kb human caudal type homeobox 2 (CDX2) promoter to delete floxed VDR alleles from the caudal region of the mouse (CDX2-KO). Weanling CDX2-KO mice and control littermates were fed low (0.25%) or normal (0.5%) Ca diets for 7 weeks. Serum and urinary Ca, vitamin D metabolites, bone parameters, and gene expression were analyzed. Loss of the VDR in CDX2-KO was confirmed in colon and kidney. Unexpectedly, CDX2-KO had lower serum PTH (–65% of controls, p < 0.001) but normal serum 1,25(OH)2D and Ca levels. Despite elevated urinary Ca loss (eightfold higher in CDX2-KO) and reduced colonic target genes TRPV6 (–90%) and CaBPD9k (–80%) mRNA levels, CDX2-KO mice had only modestly lower femoral bone density. Interestingly, duodenal TRPV6 and CaBPD9k mRNA expression was fourfold and threefold higher, respectively, and there was a trend toward increased duodenal Ca absorption (+19%, p = 0.076) in the CDX2-KO mice. The major finding of this study is that large intestine VDR significantly contributes to whole-body Ca metabolism but that duodenal compensation may prevent the consequences of VDR deletion from large intestine and kidney in growing mice. © 2015 American Society for Bone and Mineral Research. Thu, 20 Aug 2015 06:42:41 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2599 Although there has been extensive characterization of the Wnt signaling pathway in the osteoblast lineage, the effects of Wnt proteins on the osteoclast lineage are less well studied. We found that osteoclast lineage cells express canonical Wnt receptors. Wnt3a reduced osteoclast formation when applied to early bone-marrow macrophage (BMM) osteoclast differentiation cultures, whereas late addition did not suppress osteoclast formation. Early Wnt3a treatment inactivated the crucial transcription factor NFATc1 in osteoclast progenitors. Wnt3a led to the accumulation of nuclear β-catenin, confirming activation of canonical Wnt signaling. Reducing low-density lipoprotein receptor-related proteins (Lrp) 5 and Lrp6 protein expression prevented Wnt3a-induced inactivation of NFATc1; however, deletion of β-catenin did not block Wnt3a inactivation of NFATc1, suggesting that this effect was mediated by a noncanonical pathway. Wnt3a rapidly activated the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and pharmacological stimulation of cAMP/PKA signaling suppressed osteoclast differentiation; Wnt3a-induced NFATc1 phosphorylation was blocked by inhibiting interactions between PKA and A-kinase anchoring proteins (AKAPs). These data indicate that Wnt3a directly suppresses osteoclast differentiation through both canonical (β-catenin) and noncanonical (cAMP/PKA) pathways in osteoclast precursors. In vivo reduction of Lrp5 and Lrp6 expressions in the early osteoclast lineage via Rank promoter Cre recombination reduced trabecular bone mass, whereas disruption of Lrp5/6 expression in late osteoclast precursors via cathepsin K (Ctsk) promoter Cre recombination did not alter the skeletal phenotype. Surprisingly, reduction of Lrp5/6 in the early osteoclast lineage decreased osteoclast numbers, as well as osteoblast numbers. Published studies have previously noted that β-catenin signaling is required for osteoclast progenitor proliferation. Our in vivo data suggest that Rank promoter Cre-mediated deletion of Lrp5/6 may similarly impair osteoclast progenitor proliferation. © 2015 American Society for Bone and Mineral Research. Wed, 19 Aug 2015 23:53:32 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2598 A full understanding of the microenvironmental factors that control the activities of skeletal stem cells (also known as mesenchymal stem cells [MSCs]) in the adult bone marrow holds great promise for developing new therapeutic strategies to mitigate age-related diseases of bone and cartilage degeneration. Bone loss is an understudied manifestation of Marfan syndrome, a multisystem disease associated with mutations in the extracellular matrix protein and TGFβ modulator fibrillin-1. Here we demonstrate that progressive loss of cancellous bone in mice with limbs deficient for fibrillin-1 (Fbn1Prx1–/– mice) is accounted for by premature depletion of MSCs and osteoprogenitor cells combined with constitutively enhanced bone resorption. Longitudinal analyses of Fbn1Prx1–/– mice showed incremental bone loss and trabecular microarchitecture degeneration accompanied by a progressive decrease in the number and clonogenic potential of MSCs. Significant paucity of marrow fat cells in the long bones of Fbn1Prx1–/– mice, together with reduced adipogenic potential of marrow stromal cell cultures, indicated an additional defect in MSC differentiation. This postulate was corroborated by showing that an Fbn1-silenced osteoprogenitor cell line cultured in the presence of insulin yielded fewer than normal adipocytes and exhibited relatively lower PPARγ levels. Consonant with fibrillin-1 modulation of TGFβ bioavailability, cultures of marrow stromal cells from Fbn1Prx1–/– limb bones showed improper overactivation of latent TGFβ. In line with this finding, systemic TGFβ neutralization improved bone mass and trabecular microarchitecture along with normalizing the number of MSCs, osteoprogenitor cells, and marrow adipocytes. Collectively, our findings show that fibrillin-1 regulates MSC activity by modulating TGFβ bioavailability within the microenvironment of marrow niches. © 2015 American Society for Bone and Mineral Research. Mon, 17 Aug 2015 00:03:57 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2575 Longitudinal bone growth takes place in epiphyseal growth plates located in the ends of long bones. The growth plate consists of chondrocytes traversing from the undifferentiated (resting zone) to the terminally differentiated (hypertrophic zone) stage. Autophagy is an intracellular catabolic process of lysosome-dependent recycling of intracellular organelles and protein complexes. Autophagy is activated during nutritionally depleted or hypoxic conditions in order to facilitate cell survival. Chondrocytes in the middle of the growth plate are hypoxic and nutritionally depleted owing to the avascular nature of the growth plate. Accordingly, autophagy may facilitate their survival. To explore the role of autophagy in chondrocyte survival and constitutional bone growth, we generated mice with cartilage-specific ablation of either Atg5 (Atg5cKO) or Atg7 (Atg7cKO) by crossing Atg5 or Atg7 floxed mice with cartilage-specific collagen type 2 promoter–driven Cre. Both Atg5cKO and Atg7cKO mice showed growth retardation associated with enhanced chondrocyte cell death and decreased cell proliferation. Similarly, inhibition of autophagy by Bafilomycin A1 (Baf) or 3-methyladenine (3MA) promoted cell death in cultured slices of human growth plate tissue. To delineate the underlying mechanisms we employed ex vivo cultures of mouse metatarsal bones and RCJ3.IC5.18 rat chondrogenic cell line. Baf or 3MA impaired metatarsal bone growth associated with processing of caspase-3 and massive cell death. Similarly, treatment of RCJ3.IC5.18 chondrogenic cells by Baf also showed massive cell death and caspase-3 cleavage. This was associated with activation of caspase-9 and cytochrome C release. Altogether, our data suggest that autophagy is important for chondrocyte survival, and inhibition of this process leads to stunted growth and caspase-dependent death of chondrocytes. © 2015 American Society for Bone and Mineral Research. Fri, 07 Aug 2015 23:53:18 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2590 Heritable disorders that feature high bone mass (HBM) are rare. The etiology is typically a mutation(s) within a gene that regulates the differentiation and function of osteoblasts (OBs) or osteoclasts (OCs). Nevertheless, the molecular basis is unknown for approximately one-fifth of such entities. NF-κB signaling is a key regulator of bone remodeling and acts by enhancing OC survival while impairing OB maturation and function. The NF-κB transcription complex comprises five subunits. In mice, deletion of the p50 and p52 subunits together causes osteopetrosis (OPT). In humans, however, mutations within the genes that encode the NF-κB complex, including the Rela/p65 subunit, have not been reported. We describe a neonate who died suddenly and unexpectedly and was found at postmortem to have HBM documented radiographically and by skeletal histopathology. Serum was not available for study. Radiographic changes resembled malignant OPT, but histopathological investigation showed morphologically normal OCs and evidence of intact bone resorption excluding OPT. Furthermore, mutation analysis was negative for eight genes associated with OPT or HBM. Instead, accelerated bone formation appeared to account for the HBM. Subsequently, trio-based whole exome sequencing revealed a heterozygous de novo missense mutation (c.1534_1535delinsAG, p.Asp512Ser) in exon 11 of RELA encoding Rela/p65. The mutation was then verified using bidirectional Sanger sequencing. Lipopolysaccharide stimulation of patient fibroblasts elicited impaired NF-κB responses compared with healthy control fibroblasts. Five unrelated patients with unexplained HBM did not show a RELA defect. Ours is apparently the first report of a mutation within the NF-κB complex in humans. The missense change is associated with neonatal osteosclerosis from in utero increased OB function rather than failed OC action. These findings demonstrate the importance of the Rela/p65 subunit within the NF-κB pathway for human skeletal homeostasis and represent a new genetic cause of HBM. © 2015 American Society for Bone and Mineral Research. Thu, 06 Aug 2015 23:48:01 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2591 The continuously growing rodent incisor is an emerging model for the study of renewal of mineralized tissues by adult stem cells. Although the Bmp, Fgf, Shh, and Wnt pathways have been studied in this organ previously, relatively little is known about the role of Notch signaling during incisor renewal. Notch signaling components are expressed in enamel-forming ameloblasts and the underlying stratum intermedium (SI), which suggested distinct roles in incisor renewal and enamel mineralization. Here, we injected adult mice with inhibitory antibodies against several components of the Notch pathway. This blockade led to defects in the interaction between ameloblasts and the SI cells, which ultimately affected enamel formation. Furthermore, Notch signaling inhibition led to the downregulation of desmosome-specific proteins such as PERP and desmoplakin, consistent with the importance of desmosomes in the integrity of ameloblast-SI attachment and enamel formation. Together, our data demonstrate that Notch signaling is critical for proper enamel formation during incisor renewal, in part by regulating desmosome-specific components, and that the mouse incisor provides a model system to dissect Jag-Notch signaling mechanisms in the context of mineralized tissue renewal. © 2015 American Society for Bone and Mineral Research. Thu, 06 Aug 2015 11:57:30 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2584 Mitochondrial biogenesis, the generation of new mitochondrial DNA and proteins, has been linked to osteoclast (OC) differentiation and function. In this study we used mice with mutations in key alternative NF-κB pathway proteins, RelB and NF-κB–inducing kinase (NIK), to dissect the complex relationship between mitochondrial biogenesis and osteoclastogenesis. In OC precursors lacking either NIK or RelB, receptor activator of NF-κB ligand (RANKL) was unable to increase mitochondrial DNA or oxidative phosphorylation (OxPhos) protein expression, which was associated with lower oxygen consumption rates. Transgenic OC precursors expressing constitutively active NIK showed normal RANKL-induced mitochondrial biogenesis (OxPhos expression and mitochondria copy number) compared to controls, but larger mitochondrial dimensions and increased oxygen consumption rates, suggesting increased mitochondrial function. To deduce the mechanism for mitochondrial biogenesis defects in NIK-deficient and RelB-deficient precursors, we examined expression of genes known to control this process. PGC-1β (Ppargc1b) expression, but not PGC-1α, PPRC1, or ERRα, was significantly reduced in RelB–/– and NIK–/– OCs. Because PGC-1β has been reported to positively regulate both mitochondrial biogenesis and differentiation in OCs, we retrovirally overexpressed PGC-1β in RelB–/– cells, but surprisingly found that it did not affect differentiation, nor did it restore RANKL-induced mitochondrial biogenesis. To determine whether the blockade in osteoclastogenesis in RelB-deficient cells precludes mitochondrial biogenesis, we rescued RelB–/– differentiation via overexpression of NFATc1. Mitochondrial parameters in neither WT nor RelB-deficient cultures were affected by NFATc1 overexpression, and bone resorption in RelB–/– was not restored. Furthermore, NFATc1 co-overexpression with PGC-1β, although allowing OC differentiation, did not rescue mitochondrial biogenesis or bone resorption in RelB–/– OCs, by CTX-I levels. Thus, our results indicate that the alternative NF-κB pathway plays dual, but distinct, roles in controlling the independent processes of OC differentiation and OC mitochondrial biogenesis. Furthermore, the inability of PGC-1β to drive mitochondrial biogenesis in OCs without RelB indicates a cell-type specificity in mitochondria regulation. © 2015 American Society for Bone and Mineral Research. Thu, 06 Aug 2015 11:54:14 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2592 The host response to pathogens through nuclear factor κB (NF-κB) is an essential defense mechanism for eukaryotic organisms. NF-κB-mediated host responses inhibit bone and other connective tissue synthesis and are thought to affect the transcription of matrix proteins through multiple indirect pathways. We demonstrate that inhibiting NF-κB in osteoblasts increases osteocalcin expression in vivo in mice with periodontal disease. Mutating NF-κB binding sites on osteocalcin (OC) or bone sialoprotein (Bsp) promoters rescues the negative impact of NF-κB on their transcription and that NF-κB can inhibit Wnt- and Bmp-induced OC and Bsp transcription, even when protein synthesis is inhibited, indicating a direct effect of NF-κB. This inhibition depends on p65-p50 NF-κB heterodimer formation and deacetylation by HDAC1 but is not affected by the noncanonical NF-κB pathway. Moreover, NF-κB reduces Runx2 and β-catenin binding to OC/Bsp promoters independently of their nuclear localization. Thus, inflammatory signals stimulate the direct interaction of NF-κB with response elements to inhibit binding of β-catenin and Runx2 binding to nearby consensus sites and reduce expression of matrix proteins. This direct mechanism provides a new explanation for the rapid decrease in new bone formation after inflammation-related NF-κB activation. © 2015 American Society for Bone and Mineral Research. Thu, 06 Aug 2015 11:52:14 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2586 Osteoblast-mediated bone formation is coupled to osteoclast-mediated bone resorption. These processes become uncoupled with age, leading to increased risk for debilitating fractures. Therefore, understanding how osteoblasts are recruited to sites of resorption is vital to treating age-related bone loss. Osteoclasts release and activate TGF-β from the bone matrix. Here we show that osteoclast-specific inhibition of TGF-β receptor signaling in mice results in osteopenia due to reduced osteoblast numbers with no significant impact on osteoclast numbers or activity. TGF-β induced osteoclast expression of Wnt1, a protein crucial to normal bone formation, and this response was blocked by impaired TGF-β receptor signaling. Osteoclasts in aged murine bones had lower TGF-β signaling and Wnt1 expression in vivo. Ex vivo stimulation of osteoclasts derived from young or old mouse bone marrow macrophages showed no difference in TGF-β–induced Wnt1 expression. However, young osteoclasts expressed reduced Wnt1 when cultured on aged mouse bone chips compared to young mouse bone chips, consistent with decreased skeletal TGF-β availability with age. Therefore, osteoclast responses to TGF-β are essential for coupling bone resorption to bone formation, and modulating this pathway may provide opportunities to treat age-related bone loss. © 2015 American Society for Bone and Mineral Research. Thu, 06 Aug 2015 11:51:49 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2594 The association between muscle weakness and fracture is not well understood. This study sought to examine the contribution of muscle strength at baseline and change in muscle strength to the observed risk of fragility fracture in older people. The study involved 595 men and 1066 women aged 60+ years (median 69 years) who had been followed for a median of 11 years (range, 4 to 22 years). Quadriceps isometric muscle strength (MS) measured at baseline and biennially was adjusted for height. Femoral neck bone mineral density (FNBMD) was measured by DXA. Low-trauma fracture was ascertained from X-ray reports and interview. The relationship between baseline MS and serial MS and fracture assessed by time-invariant and time-variant Cox's regression models was expressed as hazard ratio (HR) and 95% confidence interval (CI). During the follow-up period, 282 (26%) women and 89 (15%) men sustained a fragility fracture. From age 60 years, women lost 0.28 kg/m (1.6%) of MS per year, whereas men lost 0.39 kg/m (1.5%) of MS per year. In the time-variant model, using serial MS, each 1 SD (4.7 kg/m) lower MS was associated with a 27% increase in the risk of fracture in women (HR 1.27; 95% CI, 1.11 to 1.43); and 46% increase in men (HR 1.46; 95% CI, 1.22 to 1.75). After adjusting for FNBMD, age and prior fracture, history of fall and smoking, HR per SD of lower MS was 1.13 (95% CI, 0.99 to 1.28) for women and 1.35 (95% CI, 1.18 to 1.64) for men. These data indicate that muscle weakness is an independent determinant of fracture risk in men, but not in women. This sex difference suggests that apart from mechanical load effect of muscle on bone, there are other muscle-bone interactions that need to be investigated in future studies. The accuracy of fracture risk prediction for men may be improved by incorporating muscle strength. © 2015 American Society for Bone and Mineral Research. Tue, 04 Aug 2015 00:11:06 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2585 Because current rat models used to study chronic kidney disease (CKD)-related vascular calcification show consistent but excessive vascular calcification and chaotic, immeasurable, bone mineralization due to excessive bone turnover, they are not suited to study the bone-vascular axis in one and the same animal. Because vascular calcification and bone mineralization are closely related to each other, an animal model in which both pathologies can be studied concomitantly is highly needed. CKD-related vascular calcification in rats was induced by a 0.25% adenine/low vitamin K diet. To follow vascular calcification and bone pathology over time, rats were killed at weeks 4, 8, 10, 11, and 12. Both static and dynamic bone parameters were measured. Vascular calcification was quantified by histomorphometry and measurement of the arterial calcium content. Stable, severe CKD was induced along with hyperphosphatemia, hypocalcemia as well as increased serum PTH and FGF23. Calcification in the aorta and peripheral arteries was present from week 8 of CKD onward. Four and 8 weeks after CKD, static and dynamic bone parameters were measurable in all animals, thereby presenting typical features of hyperparathyroid bone disease. Multiple regression analysis showed that the eroded perimeter and mineral apposition rate in the bone were strong predictors for aortic calcification. This rat model presents a stable CKD, moderate vascular calcification, and quantifiable bone pathology after 8 weeks of CKD and is the first model that lends itself to study these main complications simultaneously in CKD in mechanistic and intervention studies. © 2015 American Society for Bone and Mineral Research. Mon, 03 Aug 2015 01:31:28 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2555 Synthetic human PTH 1-34 (hPTH 1-34) replacement therapy in hypoparathyroidism maintains eucalcemia and converts quiescent bone to high-turnover bone. However, the skeletal and metabolic effects of drug discontinuation have not been reported. Nine subjects with hypoparathyroidism received subcutaneous injections of hPTH 1-34 two to three times daily for 19.8 to 61.3 months and then transitioned back to calcium and calcitriol. Biochemistries and bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA) were assessed at baseline, while on treatment, and at follow-up 3 to 12 months after drug discontinuation. Two subjects developed hypocalcemia when hPTH 1-34 was abruptly discontinued. Thus, to avoid hypocalcemia, subjects were slowly weaned from hPTH 1-34 over several weeks. When hPTH 1-34 was stopped, subjects were requiring two to three times pretreatment doses of calcitriol and calcium to maintain blood calcium levels. Doses were gradually reduced over many weeks until calcium levels were stable on doses similar to baseline. Bone-specific alkaline phosphatase (BSAP), N-telopeptide (NTX), and osteocalcin (OC) increased significantly with hPTH 1-34; at follow-up, BSAP and NTX had returned to baseline while OC was still slightly elevated. During treatment, BMD was unchanged at the hip and lateral spine but declined at the anterior-posterior (AP) spine, radius, and total body. During weaning, BMD increased, with the hip and lateral spine exceeding pre-hPTH 1-34 values and the whole body returning to baseline. AP spine was increased non-significantly compared to baseline at follow-up. hPTH 1-34 must be gradually weaned in hypoparathyroid patients with high doses of oral medications given to avoid hypocalcemia. The transient increased requirements accompanied by increased BMD after long-term hPTH 1-34 therapy suggest a reversal of the expanded remodeling space favoring bone formation as the skeleton returns to a low-turnover state, reminiscent of the hungry bone syndrome. Further study and close monitoring is required to ensure safe transition to conventional therapy and to elucidate the physiological mechanism of this phenomenon. © 2015 American Society for Bone and Mineral Research. Mon, 03 Aug 2015 01:29:50 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2589 To examine the degree of trauma in major osteoporotic fractures (MOF) in men versus women, we used data from 15,698 adults aged ≥65 years enrolled in the Osteoporotic Fractures in Men (MrOS) study (5994 men) and the Study of Osteoporotic Fractures (SOF) (9704 women). Participants were contacted tri-annually to ascertain incident fractures, which were confirmed by radiographic reports and coded according to degree of self-reported trauma. Trauma was classified as low (fall from ≤ standing height; fall on stairs, steps, or curb; minimal trauma other than fall [coughing, turning over]); moderate (collisions with objects during normal activity without associated fall); or high (fall from > standing height; severe trauma [motor vehicle accident, assault]). MOF included hip, clinical vertebral, wrist, and humerus fractures. Mean fracture follow-up was 9.1 years in SOF and 8.7 years in MrOS. A total of 14.6% of the MOF in men versus 6.3% of the MOF in women were classified as high trauma (p < 0.001); men versus women more often experienced fractures resulting from severe trauma as well as from fall > standing height. High-trauma fractures were more significantly common in men versus women at the hip (p = 0.002) and wrist (p < 0.001) but not at the spine or humerus. Among participants with MOF, the odds ratio of a fracture related to high-trauma fracture among men versus women was 3.12 (95% confidence interval [CI] 1.70–5.71) after adjustment for traditional risk factors. Findings were similar in analyses limited to participants with hip fractures (odds ratio [OR] = 3.34, 95% CI 1.04–10.67) and those with wrist fracture (OR = 5.68, 95% CI 2.03–15.85). Among community-dwelling older adults, MOF are more likely to be related to high trauma in men than in women. These findings are not explained by sex differences in conventional risk factors and may reflect a greater propensity among men to engage in risky behavior. © 2015 American Society for Bone and Mineral Research. Mon, 03 Aug 2015 01:29:13 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2582 Indian hedgehog (Ihh) is widely recognized as an essential factor for proper skeletal development. Previous in vivo studies using mutant Ihh mouse models were limited by perinatal lethality or carried out after a growth plate formed. Thus the important role of Ihh in mesenchymal cell differentiation has not been investigated. In this study, we established Prx1-Cre;Ihhfl/fl mice to ablate Ihh specifically in limb mesenchyme to allow us to observe the phenotype continuously from prenatal development to 3 weeks of age. Mutant mice displayed severe limb abnormalities characterized by complete lack of secondary ossification center and growth plate, indicating an essential role for Ihh in the development of these structures. Interestingly, we discovered that osteoblast differentiation and bone formation could occur in conditions of deficient Ihh. This is a novel finding that has not been observed because of the early lethality of previous Ihh mutants. Mature osteoblasts expressing osteocalcin could be detected in the center of mutant bones at postnatal day 10 (P10). Osteoclasts and blood vessel formation were also present, suggesting active bone remodeling. Histomorphometric analyses show a significant increase in osteoclast number with no major changes in bone formation rate at 3 weeks of age. Mutant long bones in the limbs were deformed, with cortices comprised of irregular woven bone. Also, there was a marked decrease in gene expression of osteoblastic and osteocytic markers. Moreover, mutant long bones displayed bone dysplasia in which we observed increased osteoclast activity and partially reduced osteoblastic and osteocytic differentiation that lead ultimately to loss of bone structures at 3 weeks of age. In summary, our data show for the first time, the presence of mature osteoblasts in long bones of the limbs despite the complete loss of growth plate formation due to Ihh deficiency. These data indicate an important function for Ihh in regulating limb mesenchymal cell differentiation. © 2015 American Society for Bone and Mineral Research. Wed, 29 Jul 2015 00:00:29 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2563 To investigate the role of IGF-I signaling in osterix (OSX)-expressing cells in the skeleton, we generated IGF-I receptor (IGF-IR) knockout mice (OSXIGF-IRKO) (floxed-IGF-IR mice × OSX promoter-driven GFP-labeled cre-recombinase [OSXGFPcre]), and monitored postnatal bone development. At day 2 after birth (P2), OSXGFP-cre was highly expressed in the osteoblasts in the bone surface of the metaphysis and in the prehypertrophic chondrocytes (PHCs) and inner layer of perichondral cells (IPCs). From P7, OSXGFP-cre was highly expressed in PHCs, IPCs, cartilage canals (CCs), and osteoblasts (OBs) in the epiphyseal secondary ossification center (SOC), but was only slightly expressed in the OBs in the metaphysis. Compared with the control mice, the IPC proliferation was decreased in the OSXIGF-IRKOs. In these mice, fewer IPCs invaded into the cartilage, resulting in delayed formation of the CC and SOC. Immunohistochemistry indicated a reduction of vessel number and lower expression of VEGF and ephrin B2 in the IPCs and SOC of OSXIGF-IRKOs. Quantitative real-time PCR revealed that the mRNA levels of the matrix degradation markers, MMP-9, 13 and 14, were decreased in the OSXIGF-IRKOs compared with the controls. The OSXIGF-IRKO also showed irregular morphology of the growth plate and less trabecular bone in the tibia and femur from P7 to 7 weeks, accompanied by decreased chondrocyte proliferation, altered chondrocyte differentiation, and decreased osteoblast differentiation. Our data indicate that during postnatal bone development, IGF-I signaling in OSX-expressing IPCs promotes IPC proliferation and cartilage matrix degradation and increases ephrin B2 production to stimulate vascular endothelial growth factor (VEGF) expression and vascularization. These processes are required for normal CC formation in the establishment of the SOC. Moreover, IGF-I signaling in the OSX-expressing PHC is required for growth plate maturation and osteoblast differentiation in the development of the metaphysis. © 2015 American Society for Bone and Mineral Research. Wed, 29 Jul 2015 00:00:02 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2541 Congenital orofacial abnormalities are clinically seen in human syndromes with SHP2 germline mutations such as LEOPARD and Noonan syndrome. Recent studies demonstrate that SHP2-deficiency leads to skeletal abnormalities including scoliosis and cartilaginous benign tumor metachondromatosis, suggesting that growth plate cartilage is a key tissue regulated by SHP2. The role and cellular mechanism of SHP2 in the orofacial cartilage, however, remains unknown. Here, we investigated the postnatal craniofacial development by inducible disruption of Shp2 in chondrocytes. Shp2 conditional knockout (cKO) mice displayed severe deformity of the mandibular condyle accompanied by disorganized, expanded cartilage in the trabecular bone region, enhanced type X collagen, and reduced Erk production. Interestingly, the length of primary cilia, an antenna like organelle sensing environmental signaling, was significantly shortened, and the number of primary cilia was reduced in the cKO mice. The expression levels of intraflagellar transports (IFTs), essential molecules in the assembly and function of primary cilia, were significantly decreased. Taken together, lack of Shp2 in orofacial cartilage led to severe defects of ciliogenesis through IFT reduction, resulting in mandibular condyle malformation and cartilaginous expansion. Our study provides new insights into the molecular pathogenesis of SHP2-deficiency in cartilage and helps to understand orofacial and skeletal manifestations seen in patients with SHP2 mutations. © 2015 American Society for Bone and Mineral Research. Tue, 28 Jul 2015 23:59:15 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2554 The acid load accompanying modern diets may have adverse effects on bone and muscle metabolism. Treatment with alkaline salts of potassium can neutralize the acid load, but the optimal amount of alkali is not established. Our objective was to determine the effectiveness of two doses of potassium bicarbonate (KHCO3) compared with placebo on biochemical markers of bone turnover, and calcium and nitrogen (N) excretion. In this double-blind, randomized, placebo-controlled study, 244 men and women age 50 years and older were randomized to placebo or 1 mmol/kg or 1.5 mmol/kg of KHCO3 daily for 3 months; 233 completed the study. The primary outcomes were changes in 24-hour urinary N-telopeptide (NTX) and N; changes in these measures were compared across the treatment groups. Exploratory outcomes included 24-hour urinary calcium excretion, serum amino-terminal propeptide of type I procollagen (P1NP), and muscle strength and function assessments. The median administered doses in the low-dose and high-dose groups were 81 mmol/day and 122 mmol/day, respectively. When compared with placebo, urinary NTX declined significantly in the low-dose group (p = 0.012, after adjustment for baseline NTX, gender, and change in urine creatinine) and serum P1NP declined significantly in the low-dose group (p = 0.004, adjusted for baseline P1NP and gender). Urinary calcium declined significantly in both KHCO3 groups versus placebo (p < 0.001, adjusted for baseline urinary calcium, gender, and changes in urine creatinine and calcium intake). There was no significant effect of either dose of KHCO3 on urinary N excretion or on the physical strength and function measures. KHCO3 has favorable effects on bone turnover and calcium excretion and the lower dose appears to be the more effective dose. Long-term trials to assess the effect of alkali on bone mass and fracture risk are needed. © 2015 American Society for Bone and Mineral Research. Tue, 28 Jul 2015 23:58:50 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2588 Patients with hypoparathyroidism have low circulating parathyroid (PTH) levels and higher cancellous bone volume and trabecular thickness. Treatment with PTH(1-84) was shown to increase abnormally low bone remodeling dynamics. In this work, we studied the effect of 1-year or 2-year PTH(1-84) treatment on cancellous and cortical bone mineralization density distribution (Cn.BMDD and Ct.BMDD) based on quantitative backscattered electron imaging (qBEI) in paired transiliac bone biopsy samples. The study cohort comprised 30 adult hypoparathyroid patients (14 treated for 1 year; 16 treated for 2 years). At baseline, Cn.BMDD was shifted to higher mineralization densities in both treatment groups (average degree of mineralization Cn.CaMean +3.9% and +2.7%, p < 0.001) compared to reference BMDD. After 1-year PTH(1-84), Cn.CaMean was significantly lower than that at baseline (–6.3%, p < 0.001), whereas in the 2-year PTH(1-84) group Cn.CaMean did not differ from baseline. Significant changes of Ct.BMDD were observed in the 1-year treatment group only. The change in histomorphometric bone formation (mineralizing surface) was predictive for Cn.BMDD outcomes in the 1-year PTH(1-84) group, but not in the 2-year PTH(1-84) group. Our findings suggest higher baseline bone matrix mineralization consistent with the decreased bone turnover in hypoparathyroidism. PTH(1-84) treatment caused differential effects dependent on treatment duration that were consistent with the histomorphometric bone formation outcomes. The greater increase in bone formation during the first year of treatment was associated with a decrease in bone matrix mineralization, suggesting that PTH(1-84) exposure to the hypoparathyroid skeleton has the greatest effects on BMDD early in treatment. © 2015 American Society for Bone and Mineral Research. Tue, 28 Jul 2015 23:58:00 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2542 Vascular hyperpermeability and highly upregulated bone resorption in the destructive repair progress of steroid-associated osteonecrosis (SAON) are associated with a high expression of VEGF and high Src activity (Src is encoded by the cellular sarcoma [c-src] gene). This study was designed to prove our hypothesis that blocking the VEGF-Src signaling pathway by specific Src siRNA is able to prevent destructive repair in a SAON rabbit model. Destructive repair in SAON was induced in rabbits. At 2, 4, and 6 weeks after SAON induction, VEGF, anti-VEGF, Src siRNA, Src siRNA+VEGF, control siRNA, and saline were introduced via intramedullary injection into proximal femora for each group, respectively. Vascularization and permeability were quantified by dynamic contrast-enhanced (DCE) MRI. At week 6 after SAON induction, proximal femurs were dissected for micro–computed tomography (μCT)-based trabecular architecture with finite element analysis (FEA), μCT-based angiography, and histological analysis. Histological evaluation revealed that VEGF enhanced destructive repair, whereas anti-VEGF prevented destructive repair and Src siRNA and Src siRNA+VEGF prevented destructive repair and enhanced reparative osteogenesis. Findings of angiography and histomorphometry were consistent with those determined by DCE MRI. Src siRNA inhibited VEGF-mediated vascular hyperpermeability but preserved VEGF-induced neovascularization. Bone resorption was enhanced in the VEGF group and inhibited in the anti-VEGF, Src siRNA, Src siRNA+VEGF groups as determined by both 3D μCT and 2D histomorphometry. FEA showed higher estimated failure load in the Src siRNA and Src siRNA+VEGF groups when compared to the vehicle control group. Blockage of VEGF-Src signaling pathway by specific Src siRNA was able to prevent steroid-associated destructive repair while improving reconstructive repair in SAON, which might become a novel therapeutic strategy. © 2015 American Society for Bone and Mineral Research. Fri, 24 Jul 2015 07:35:59 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2581 Cells of the osteoblast lineage provide critical support for B lymphopoiesis in the bone marrow (BM). Parathyroid hormone (PTH) signaling in osteoblastic cells through its receptor (PPR) is an important regulator of hematopoietic stem cells; however, its role in regulation of B lymphopoiesis is not clear. Here we demonstrate that deletion of PPR in osteoprogenitors results in a significant loss of trabecular and cortical bone. PPR signaling in osteoprogenitors, but not in mature osteoblasts or osteocytes, is critical for B-cell precursor differentiation via IL-7 production. Interestingly, despite a severe reduction in B-cell progenitors in BM, mature B-lymphocytes were increased 3.5-fold in the BM of mice lacking PPR in osteoprogenitors. This retention of mature IgD+ B cells in the BM was associated with increased expression of vascular cell adhesion molecule 1 (VCAM1) by PPR-deficient osteoprogenitors, and treatment with VCAM1 neutralizing antibody increased mobilization of B lymphocytes from mutant BM. Our results demonstrate that PPR signaling in early osteoblasts is necessary for B-cell differentiation via IL-7 secretion and for B-lymphocyte mobilization via VCAM1. © 2015 American Society for Bone and Mineral Research. Mon, 20 Jul 2015 07:06:42 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2573 The primary goal of this cross-sectional in vivo study was to assess peripheral bone microarchitecture, bone strength, and bone remodeling in adult type 1 diabetes (T1D) patients with and without diabetic microvascular disease (MVD+ and MVD–, respectively) and to compare them with age-, gender-, and height-matched healthy control subjects (CoMVD+ and CoMVD–, respectively). The secondary goal was to assess differences in MVD– and MVD+ patients. Fifty-five patients with T1DM (MVD+ group: n = 29) were recruited from the Funen Diabetes Database. Dual-energy X-ray absorptiometry (DXA), high-resolution peripheral quantitative computed tomography (HR-pQCT) of the ultradistal radius and tibia, and biochemical markers of bone turnover were performed in all participants. There were no significant differences in HR-pQCT parameters between MVD– and CoMVD– subjects. In contrast, MVD+ patients had larger total and trabecular bone areas (p = 0.04 and p = 0.02, respectively), lower total, trabecular, and cortical volumetric bone mineral density (vBMD) (p < 0.01, p < 0.04, and p < 0.02, respectively), and thinner cortex (p = 0.03) at the radius, and lower total and trabecular vBMD (p = 0.01 and p = 0.02, respectively) at the tibia in comparison to CoMVD+. MVD+ patients also exhibited lower total and trabecular vBMD (radius p = 0.01, tibia p < 0.01), trabecular thickness (radius p = 0.01), estimated bone strength, and greater trabecular separation (radius p = 0.01, tibia p < 0.01) and network inhomogeneity (radius p = 0.01, tibia p < 0.01) in comparison to MVD– patients. These differences remained significant after adjustment for age, body mass index, gender, disease duration, and glycemic control (average glycated hemoglobin over the previous 3 years). Although biochemical markers of bone turnover were significantly lower in MVD+ and MVD– groups in comparison to controls, they were similar between the MVD+ and MVD– groups. The results of our study suggest that the presence of MVD was associated with deficits in cortical and trabecular bone vBMD and microarchitecture that could partly explain the excess skeletal fragility observed in these patients. © 2015 American Society for Bone and Mineral Research. Mon, 20 Jul 2015 07:04:13 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2551 Activating mutations of calcium-sensing receptor (CaSR) cause autosomal dominant hypocalcemia (ADH). ADH patients develop hypocalcemia, hyperphosphatemia, and hypercalciuria, similar to the clinical features of hypoparathyroidism. The current treatment of ADH is similar to the other forms of hypoparathyroidism, using active vitamin D3 or parathyroid hormone (PTH). However, these treatments aggravate hypercalciuria and renal calcification. Thus, new therapeutic strategies for ADH are needed. Calcilytics are allosteric antagonists of CaSR, and may be effective for the treatment of ADH caused by activating mutations of CaSR. In order to examine the effect of calcilytic JTT-305/MK-5442 on CaSR harboring activating mutations in the extracellular and transmembrane domains in vitro, we first transfected a mutated CaSR gene into HEK cells. JTT-305/MK-5442 suppressed the hypersensitivity to extracellular Ca2+ of HEK cells transfected with the CaSR gene with activating mutations in the extracellular and transmembrane domains. We then selected two activating mutations locating in the extracellular (C129S) and transmembrane (A843E) domains, and generated two strains of CaSR knock-in mice to build an ADH mouse model. Both mutant mice mimicked almost all the clinical features of human ADH. JTT-305/MK-5442 treatment in vivo increased urinary cAMP excretion, improved serum and urinary calcium and phosphate levels by stimulating endogenous PTH secretion, and prevented renal calcification. In contrast, PTH(1-34) treatment normalized serum calcium and phosphate but could not reduce hypercalciuria or renal calcification. CaSR knock-in mice exhibited low bone turnover due to the deficiency of PTH, and JTT-305/MK-5442 as well as PTH(1-34) increased bone turnover and bone mineral density (BMD) in these mice. These results demonstrate that calcilytics can reverse almost all the phenotypes of ADH including hypercalciuria and renal calcification, and suggest that calcilytics can become a novel therapeutic agent for ADH. © 2015 American Society for Bone and Mineral Research. Thu, 16 Jul 2015 23:51:36 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2556 Teriparatide, a recombinant peptide corresponding to amino acids 1-34 of human parathyroid hormone (PTH), has been an effective bone anabolic drug for over a decade. However, the mechanism whereby PTH stimulates bone formation remains incompletely understood. Here we report that in cultures of osteoblast-lineage cells, PTH stimulates glucose consumption and lactate production in the presence of oxygen, a hallmark of aerobic glycolysis, also known as Warburg effect. Experiments with radioactively labeled glucose demonstrate that PTH suppresses glucose entry into the tricarboxylic acid cycle (TCA cycle). Mechanistically, the increase in aerobic glycolysis is secondary to insulin-like growth factor (Igf) signaling induced by PTH, whereas the metabolic effect of Igf is dependent on activation of mammalian target of rapamycin complex 2 (mTORC2). Importantly, pharmacological perturbation of glycolysis suppresses the bone anabolic effect of intermittent PTH in the mouse. Thus, stimulation of aerobic glycolysis via Igf signaling contributes to bone anabolism in response to PTH. © 2015 American Society for Bone and Mineral Research. Tue, 14 Jul 2015 23:53:41 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2557 Bone marrow fat, an unique component of the bone marrow cavity increases with aging and menopause and is inversely related to bone mass. Sex steroids may be involved in the regulation of bone marrow fat, because men have higher bone marrow fat than women and clinical observations have suggested that the variation in bone marrow fat fraction is greater in premenopausal compared to postmenopausal women and men. We hypothesized that the menstrual cycle and/or estrogen affects the bone marrow fat fraction. First, we measured vertebral bone marrow fat fraction with Dixon Quantitative Chemical Shift MRI (QCSI) twice a week during 1 month in 10 regularly ovulating women. The vertebral bone marrow fat fraction increased 0.02 (95% CI, 0.00 to 0.03) during the follicular phase (p = 0.033), and showed a nonsignificant decrease of 0.02 (95% CI, –0.01 to 0.04) during the luteal phase (p = 0.091). To determine the effect of estrogen on bone marrow fat, we measured vertebral bone marrow fat fraction every week for 6 consecutive weeks in 6 postmenopausal women before, during, and after 2 weeks of oral 17-β estradiol treatment (2 mg/day). Bone marrow fat fraction decreased by 0.05 (95% CI, 0.01 to 0.09) from 0.48 (95% CI, 0.42 to 0.53) to 0.43 (95% CI, 0.34 to 0.51) during 17-β estradiol administration (p < 0.001) and increased again after cessation. During 17-β estradiol administration the bone formation marker procollagen type I N propeptide (P1NP) increased (p = 0.034) and the bone resorption marker C-terminal crosslinking telopeptides of collagen type I (CTx) decreased (p < 0.001). In conclusion, we described the variation in vertebral bone marrow fat fraction among ovulating premenopausal women. And among postmenopausal women, we demonstrated that 17-β estradiol rapidly reduces the marrow fat fraction, suggesting that 17-β estradiol regulates bone marrow fat independent of bone mass. © 2015 American Society for Bone and Mineral Research. Tue, 14 Jul 2015 23:53:09 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2572 Although a large number of studies have addressed the age-related changes in bone mineral density (BMD), there is a paucity of data for the assessment of femoral strength loss with age in both genders. We determined the variation of strength with age in femurs of women and men by mechanical tests on a cohort of 100 cadaveric femurs. In addition, the age-related neck BMD loss in our cadaveric cohort was found to be similar with BMD loss of four published population-based studies. Given the strong correlation found in our cadaveric study between BMD and femoral strength, we also estimated the femoral strength of the four populations based on their reported neck BMDs. Our study showed that men's femurs in our cadaveric cohort were stronger than women's femurs by about 800 N at the same BMD level, and by 1750 N at the same age. The strength differences were not explained satisfactorily by the size difference between men's and women's bones. Similar to the findings of clinical studies, the BMD values of men at all ages were larger than that of women. The age-related loss rates in BMD and strength were not statistically different between the two genders of our cadaveric cohort. After normalization, strength decreased more than 40% faster than BMD. On average, men reached a certain BMD value about 16 years later than women, and for strength about 23 years later, which may explain the higher rate of hip fracture in postmenopausal women. In patient population cohorts men reached a similar BMD value about 16 to 25 years later than women, whereas for estimated strength, sometimes more than 40 years later. © 2015 American Society for Bone and Mineral Research. Tue, 14 Jul 2015 23:52:09 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2580 Magnetic resonance has the potential to image and quantify two pools of water within bone: free water within the Haversian pore system (transverse relaxation time, T2> 1 ms), and water hydrogen-bonded to matrix collagen (T2 ∼ 300 to 400 μs). Although total bone water concentration quantified by MRI has been shown to scale with porosity, greater insight into bone matrix density and porosity may be gained by relaxation-based separation of bound and pore water fractions. The objective of this study was to evaluate a recently developed surrogate measurement for matrix density, single adiabatic inversion recovery (SIR) zero echo-time (ZTE) MRI, in human bone. Specimens of tibial cortical bone from 15 donors (aged 27 to 97 years; 8 female and 7 male) were examined at 9.4T field strength using two methods: (1) 1H ZTE MRI, to capture total 1H signal, and (2) 1H SIR-ZTE MRI, to selectively image matrix-associated 1H signal. Total water, bone matrix, and bone mineral densities were also quantified gravimetrically, and porosity was measured by micro-CT. ZTE apparent total water 1H concentration was 32.7 ± 3.2 M (range 28.5 to 40.3 M), and was correlated positively with porosity (R2 = 0.80) and negatively with matrix and mineral densities (R2 =  0.90 and 0.82, respectively). SIR-ZTE apparent bound water 1H concentration was 32.9 ± 3.9 M (range 24.4 to 39.8 M), and its correlations were opposite to those of apparent total water: negative with porosity (R2 = 0.73) and positive with matrix density (R2 = 0.74) and mineral density (R2 = 0.72). Porosity was strongly correlated with gravimetric matrix density (R2 = 0.91, negative) and total water density (R2 = 0.92, positive). The strong correlations of SIR-ZTE-derived apparent bound water 1H concentration with ground-truth measurements suggest that this quantitative solid-state MRI method provides a nondestructive surrogate measure of bone matrix density. © 2015 American Society for Bone and Mineral Research. Tue, 14 Jul 2015 23:51:50 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2570 We revisit Stanley Garn's theory related to sex differences in endocortical and periosteal apposition during adolescence using a 12-year mixed longitudinal study design. We used peripheral quantitative computed tomography to examine bone parameters in 230 participants (110 boys, 120 girls; aged 11.0 years at baseline). We assessed total (Tt.Ar, mm2), cortical (Ct.Ar, mm2), and medullary canal area (Me.Ar, mm2), Ct.Ar/Tt.Ar, cortical bone mineral density (Ct.BMD, mg/cm3), and polar strength-strain index (SSIp, mm3) at the tibial midshaft (50% site). We used annual measures of height and chronological age to identify age at peak height velocity (APHV) for each participant. We compared annual accrual rates of bone parameters between boys and girls, aligned on APHV using a linear mixed effects model. At APHV, boys demonstrated greater Tt.Ar (ratio = 1.27; 95% confidence interval [CI] 1.21, 1.32), Ct.Ar (1.24 [1.18, 1.30]), Me.Ar (1.31 [1.22, 1.40]), and SSIp (1.36 [1.28, 1.45]) and less Ct.Ar/Tt.Ar (0.98 [0.96, 1.00]) and Ct.BMD (0.97 [0.96, 0.97]) compared with girls. Boys and girls demonstrated periosteal bone formation and net bone loss at the endocortical surface. Compared with girls, boys demonstrated greater annual accrual rates pre-APHV for Tt.Ar (1.18 [1.02, 1.34]) and Me.Ar (1.34 [1.11, 1.57]), lower annual accrual rates pre-APHV for Ct.Ar/Tt.Ar (0.56 [0.29, 0.83]) and Ct.BMD (–0.07 [–0.17, 0.04]), and similar annual accrual rates pre-APHV for Ct.Ar (1.10 [0.94, 1.26]) and SSIp (1.14 [0.98, 1.30]). Post-APHV, boys demonstrated similar annual accrual rates for Ct.Ar/Tt.Ar (1.01 [0.71, 1.31]) and greater annual accrual rates for all other bone parameters compared with girls (ratio = 1.23 to 2.63; 95% CI 1.11 to 3.45). Our findings support those of Garn and others of accelerated periosteal apposition during adolescence, more evident in boys than girls. However, our findings challenge the notion of greater endocortical apposition in girls, suggesting instead that girls experience diminished endocortical resorption compared with boys. © 2015 American Society for Bone and Mineral Research. Tue, 14 Jul 2015 09:29:47 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2579 Hip fractures are associated with high excess mortality. Education is an important determinant of health, but little is known about educational inequalities in post-hip fracture mortality. Our objective was to investigate educational inequalities in post-hip fracture mortality and to examine whether comorbidity or family composition could explain any association. We conducted a register-based population study of Norwegians aged 50 years and older from 2002 to 2010. We measured total mortality according to educational attainment in 56,269 hip fracture patients (NORHip) and in the general Norwegian population. Both absolute and relative educational inequalities in mortality in people with and without hip fracture were compared. There was an educational gradient in post-hip fracture mortality in both sexes. Compared with those with primary education only, the age-adjusted relative risk (RR) of mortality in hip fracture patients with tertiary education was 0.82 (95% confidence interval [CI] 0.77–0.87) in men and 0.79 (95% CI 0.75–0.84) in women. Additional adjustments for Charlson comorbidity index, marital status, and number of children did not materially change the estimates. Regardless of educational attainment, the 1-year age-adjusted mortality was three- to fivefold higher in hip fracture patients compared with peers in the general population without fracture. The absolute differences in 1-year mortality according to educational attainment were considerably larger in hip fracture patients than in the population without hip fracture. Absolute educational inequalities in mortality were higher after hip fracture compared with the general population without hip fracture and were not mediated by comorbidity or family composition. Investigation of other possible mediating factors might help to identify new targets for interventions, based on lower educational attainment, to reduce post-hip fracture mortality. © 2015 American Society for Bone and Mineral Research. Tue, 14 Jul 2015 09:29:38 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2577 Diabetes mellitus is an established risk factor associated with bisphosphonate-related osteonecrosis of the jaw (BRONJ). Sustained activation of Nod-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome contributes to the persistent inflammation and impaired cutaneous wound healing in diabetic mice and human. We have recently demonstrated a compelling linkage between M1 macrophages and BRONJ conditions in both murine and human diseases. The aim of this study was to determine whether NLRP3 inflammasome activation is involved in BRONJ development in diabetic mice. We showed an increased incidence of delayed oral wound healing and bone necrosis of extraction sockets in db/db mice compared with those in nondiabetic db/+ controls, which correlated with an elevated expression of NLRP3, caspase-1, and IL-1β in macrophages residing at local wounds. Constitutively, bone marrow-derived macrophages from db/db mice (db/db BMDMs) secrete a relatively higher level of IL-1β than those from db/+ mice (db/+ BMDMs). Upon stimulation by NLRP3 activators, the secretion of IL-1β by db/db BMDMs was 1.77-fold higher than that by db/+ BMDMs (p < 0.001). Systemic treatment of mice with zoledronate (Zol), a nitrogen-containing bisphosphonate, resulted in a 1.86- and 1.63-fold increase in NLRP3/caspase-1-dependent IL-1β secretion by db/+ and db/db BMDMs, respectively, compared with BMDMs derived from nontreated mice (p < 0.001). Importantly, systemic administration of pharmacological inhibitors of NLRP3 activation improved oral wound healing and suppressed BRONJ formation in db/db mice. Mechanistically, we showed that supplementation with intermediate metabolites of the mevalonate pathway, inhibitors of caspase-1 and NLRP3 activation, an antagonist for P2X7R, or a scavenger of reactive oxygen species (ROS), robustly abolished Zol-enhanced IL-1β release from macrophages in response to NLRP3 activation (p < 0.001). Our findings suggest that diabetes-associated chronic inflammatory response may have contributed to impaired socket wound healing and rendered oral wound susceptible to the development of BRONJ via NLRP3 activation in macrophages. © 2015 American Society for Bone and Mineral Research. Tue, 14 Jul 2015 08:22:27 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2552 Hip fracture risk is known to be related to material properties of the proximal femur, but fracture prediction studies adding richer quantitative computed tomography (QCT) measures to dual-energy X-ray (DXA)-based methods have shown limited improvement. Fracture types have distinct relationships to predictors, but few studies have subdivided fracture into types, because this necessitates regional measurements and more fracture cases. This work makes use of cortical bone mapping (CBM) to accurately assess, with no prior anatomical presumptions, the distribution of properties related to fracture type. CBM uses QCT data to measure the cortical and trabecular properties, accurate even for thin cortices below the imaging resolution. The Osteoporotic Fractures in Men (MrOS) study is a predictive case-cohort study of men over 65 years old: we analyze 99 fracture cases (44 trochanteric and 55 femoral neck) compared to a cohort of 308, randomly selected from 5994. To our knowledge, this is the largest QCT-based predictive hip fracture study to date, and the first to incorporate CBM analysis into fracture prediction. We show that both cortical mass surface density and endocortical trabecular BMD are significantly different in fracture cases versus cohort, in regions appropriate to fracture type. We incorporate these regions into predictive models using Cox proportional hazards regression to estimate hazard ratios, and logistic regression to estimate area under the receiver operating characteristic curve (AUC). Adding CBM to DXA-based BMD leads to a small but significant (p < 0.005) improvement in model prediction for any fracture, with AUC increasing from 0.78 to 0.79, assessed using leave-one-out cross-validation. For specific fracture types, the improvement is more significant (p < 0.0001), with AUC increasing from 0.71 to 0.77 for trochanteric fractures and 0.76 to 0.82 for femoral neck fractures. In contrast, adding DXA-based BMD to a CBM-based predictive model does not result in any significant improvement. © 2015 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research. Tue, 14 Jul 2015 08:22:11 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2568 The diagnosis of fracture risk relies almost solely on quantifying bone mass, yet bone strength is governed by factors at multiple scales including composition and structure that contribute to fracture resistance. Furthermore, aging and conditions such as diabetes mellitus alter fracture incidence independently of bone mass. Therefore, it is critical to incorporate other factors that contribute to bone strength in order to improve diagnostic specificity of fracture risk. We examined the correlation between femoral neck fracture strength in aging female cadavers and areal bone mineral density, along with other clinically accessible measures of bone quality including whole-bone cortical porosity (Ct.Po), bone material mechanical behavior measured by reference point indentation (RPI), and accumulation of advanced glycation end-products (AGEs). All measurements were found to be significant predictors of femoral neck fracture strength, with areal bone mineral density (aBMD) being the single strongest correlate (aBMD: r = 0.755, p < 0.001; Ct.Po: r = –0.500, p < 0.001; RPI: r = –0.478, p < 0.001; AGEs: r = –0.336, p = 0.016). RPI-derived measurements were not correlated with tissue mineral density or local cortical porosity as confirmed by micro–computed tomography (μCT). Multiple reverse stepwise regression revealed that the inclusion of aBMD and any other factor significantly improve the prediction of bone strength over univariate predictions. Combining bone assays at multiple scales such as aBMD with tibial Ct.Po (r = 0.835; p < 0.001), tibial difference in indentation depth between the first and 20th cycle (IDI) (r = 0.883; p < 0.001), or tibial AGEs (r = 0.822; p < 0.001) significantly improves the prediction of femoral neck strength over any factor alone, suggesting that this personalized approach could greatly enhance bone strength and fracture risk assessment with the potential to guide clinical management strategies for at-risk populations. © 2015 American Society for Bone and Mineral Research. Tue, 14 Jul 2015 08:21:56 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2337 Cover: Scanning electron micrograph of an acid-activated mouse osteoclast with resorption trail. Image width, 0.1 mm. Contributed by Tim Arnett, Mark Turmaine and Javier Manzano, University College London. Tue, 22 Sep 2015 04:18:09 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2531 A position paper of the National Bone Health Alliance recently recommended that diagnostic criteria for osteoporosis be redefined. We review the merits and demerits of this proposal and argue that the operational bone mineral density (BMD)-based definition be retained while clarity is brought to bear on the distinction between diagnostic and intervention thresholds. © 2015 American Society for Bone and Mineral Research. Tue, 22 Sep 2015 04:18:06 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2686 Thu, 03 Sep 2015 01:24:52 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2515 The aim of this population-based, prospective cohort study was to investigate long-term associations between dietary calcium intake and fractures, non-fatal cardiovascular disease (CVD), and death from all causes. Participants were from the Melbourne Collaborative Cohort Study, which was established in 1990 to 1994. A total of 41,514 men and women (∼99% aged 40 to 69 years at baseline) were followed up for a mean (SD) of 12 (1.5) years. Primary outcome measures were time to death from all causes (n = 2855), CVD-related deaths (n = 557), cerebrovascular disease-related deaths (n = 139), incident non-fatal CVD (n = 1827), incident stroke events (n = 537), and incident fractures (n = 788). A total of 12,097 participants (aged ≥50 years) were eligible for fracture analysis and 34,468 for non-fatal CVD and mortality analyses. Mortality was ascertained by record linkage to registries. Fractures and CVD were ascertained from interview ∼13 years after baseline. Quartiles of baseline energy-adjusted calcium intake from food were estimated using a food-frequency questionnaire. Hazard ratios (HR) and odds ratios (OR) were calculated for quartiles of dietary calcium intake. Highest and lowest quartiles of energy-adjusted dietary calcium intakes represented unadjusted means (SD) of 1348 (316) mg/d and 473 (91) mg/d, respectively. Overall, there were 788 (10.3%) incident fractures, 1827 (9.0%) incident CVD, and 2855 people (8.6%) died. Comparing the highest with the lowest quartile of calcium intake, for all-cause mortality, the HR was 0.86 (95% confidence interval [CI] 0.76–0.98, ptrend = 0.01); for non-fatal CVD and stroke, the OR was 0.84 (95% CI 0.70–0.99, ptrend = 0.04) and 0.69 (95% CI 0.51–0.93, ptrend = 0.02), respectively; and the OR for fracture was 0.70 (95% CI 0.54–0.92, ptrend = 0.004). In summary, for older men and women, calcium intakes of up to 1348 (316) mg/d from food were associated with decreased risks for fracture, non-fatal CVD, stroke, and all-cause mortality. © 2015 American Society for Bone and Mineral Research. Sun, 10 May 2015 23:27:54 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2530 There is increasing evidence that bone and vascular calcification share common pathogenesis. Little is known about potential links between bone and valvular calcification. The purpose of this study was to determine the association between spine bone mineral density (BMD) and vascular and valvular calcification. Participants included 1317 participants (689 women, 628 men) in the Framingham Offspring Study (mean age 60 years). Integral, trabecular, and cortical volumetric bone density (vBMD) and arterial and valvular calcification were measured from computed tomography (CT) scans and categorized by sex-specific quartiles (Q4 = high vBMD). Calcification of the coronary arteries (CAC), abdominal aorta (AAC), aortic valve (AVC), and mitral valve (MVC) were quantified using the Agatston Score (AS). Prevalence of any calcium (AS >0) was 69% for CAC, 81% for AAC, 39% for AVC, and 20% for MVC. In women, CAC increased with decreasing quartile of trabecular vBMD: adjusted mean CAC = 2.1 (Q4), 2.2 (Q3), 2.5 (Q2), 2.6 (Q1); trend p = 0.04. However, there was no inverse trend between CAC and trabecular vBMD in men: CAC = 4.3 (Q4), 4.3 (Q3), 4.2 (Q2), 4.3 (Q1); trend p = 0.92. AAC increased with decreasing quartile of trabecular vBMD in both women (AAC = 4.5 [Q4], 4.8 [Q3], 5.4 [Q2], 5.1 [Q1]; trend p = 0.01) and men (AAC = 5.5 [Q4], 5.8 [Q3], 5.9 [Q2], 6.2 [Q1]; trend p = 0.01). We observed no association between trabecular vBMD and AVC or MVC in women or men. Finally, cortical vBMD was unrelated to vascular calcification and valvular calcification in women and men. Women and men with low spine vBMD have greater severity of vascular calcification, particularly at the abdominal aorta. The inverse relation between AAC and spine vBMD in women and men may be attributable to shared etiology and may be an important link on which to focus treatment efforts that can target individuals at high risk of both fracture and cardiovascular events. © 2015 American Society for Bone and Mineral Research. Wed, 06 May 2015 10:05:14 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2521 Psoriatic arthritis (PsA) is a chronic inflammatory disease characterized by periarticular bone loss and new bone formation. Current data regarding systemic bone loss and bone mineral density (BMD) in PsA are conflicting. The aim of this study was to evaluate bone microstructure and volumetric BMD (vBMD) in patients with PsA and psoriasis. We performed HR-pQCT scans at the ultradistal and periarticular radius in 50 PsA patients, 30 psoriasis patients, and 70 healthy, age- and sex-related controls assessing trabecular bone volume (BV/TV), trabecular number (Tb.N), inhomogeneity of the trabecular network, cortical thickness (Ct.Th), and cortical porosity (Ct.Po), as well as vBMD. Trabecular BMD (Tb.BMD, p = 0.021, 12.0%), BV/TV (p = 0.020, –11.9%), and Tb.N (p = 0.035, 7.1%) were significantly decreased at the ultradistal radius and the periarticular radius in PsA patients compared to controls. In contrast, bone architecture of the ultradistal radius and periarticular radius was similar in patients with psoriasis and healthy controls. Duration of skin disease was associated with low BV/TV and Tb.N in patients with PsA. These data suggest that trabecular BMD and bone microstructure are decreased in PsA patients. The observation that duration of skin disease determines bone loss in PsA supports the concept of subclinical musculoskeletal disease in psoriasis patients. © 2015 American Society for Bone and Mineral Research. Sun, 10 May 2015 23:28:09 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2537 Hip fracture rates in Norway rank among the highest in the world, more than double that of Spanish women. Previous studies were unable to demonstrate significant differences between the two populations with respect to bone mass or calcium metabolism. In order to test whether the difference in fracture propensity between both populations could be explained by differences in bone material quality we assessed bone material strength using microindentation in 42 Norwegian and 46 Spanish women with normal BMD values, without clinical or morphometric vertebral fractures, no clinical or laboratory signs of secondary osteoporosis, and without use of drugs with known influence on bone metabolism. Bone material properties were assessed by microindentation of the thick cortex of the mid tibia following local anesthesia of the area using the Osteoprobe device (Active Life Scientific, Santa Barbara, CA, USA). Indentation distance was standardized against a calibration phantom of methylmethacrylate and results, as percentage of this reference value, expressed as bone material strength index units (BMSi). We found that the bone material properties reflected in the BMSi value of Norwegian women was significantly inferior when compared to Spanish women (77 ± 7.1 versus 80.7 ± 7.8, p < 0.001). Total hip BMD was significantly higher in Norwegian women (1.218 g/cm2 versus 0.938 g/cm2, p < 0.001) but regression analysis revealed that indentation values did not vary with BMD r2 = 0.03 or age r2 = 0.04. In conclusion Norwegian women show impaired bone material properties, higher bone mass, and were taller than Spanish women. The increased height will increase the impact on bone after falls, and impaired bone material properties may further enhance the risk fracture after such falls. These ethnic differences in bone material properties may partly explain the higher propensity for fracture in Norwegian women. © 2015 American Society for Bone and Mineral Research. Thu, 21 May 2015 05:03:16 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2519 Radiotherapy, an essential component of cancer treatment, is not without risk to bone, particularly to the immature or growing skeleton. Known side effects range from post-radiation osteitis to osteoradionecrosis. We report the case of a 14-year-old male patient undergoing denosumab treatment, a new antiresorptive agent, for osteoradionecrosis. The patient exhibited fractures and associated pain and functional limitations secondary to radiation for the treatment of an embryonal rhabdomyosarcoma of prostate grade III administered at age 5 years. After treatment with denosumab, the pain disappeared, bone remodeling markers dramatically declined, bone mass increased, and pathological bone scan findings resolved without adverse effects or new fractures. © 2015 American Society for Bone and Mineral Research. Thu, 21 May 2015 05:04:10 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2516 Neonatal severe hyperparathyroidism (NSHPT) is a rare disorder characterized by major hypercalcemia, elevated parathyroid hormone levels, and marked enlargement of multiple parathyroid glands, usually associated with germline mutations in the calcium receptor gene CASR. However, little is known about the outgrowth of parathyroid tumors in NSHPT, including whether they represent monoclonal or polyclonal expansions. We sought to examine the clonality of parathyroid tissues resected from a patient with NSHPT and biallelic CASR mutations. DNA from two distinct parathyroid tumors resected from a girl with NSHPT, plus polyclonal/monoclonal control samples, were subjected to analyses of clonality by two independent methods, X-chromosome inactivation analysis at the androgen receptor locus (HUMARA) and BAC array comparative genomic hybridization (CGH). Both parathyroid tumor samples revealed polyclonal patterns by X-inactivation analysis, with polyclonal and monoclonal controls yielding the expected patterns. Similarly, by BAC array CGH, neither parathyroid sample contained monoclonal copy number changes and both appeared identical to the patient-matched polyclonal controls. Our observations provide direct experimental evidence that the markedly enlarged parathyroid tumors in the setting of NSHPT constitute polyclonal, generalized hyperplastic growths rather than monoclonal neoplasms. © 2015 American Society for Bone and Mineral Research. Thu, 14 May 2015 09:32:28 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2532 Hypocalcemia and hyperphosphatemia are encountered in idiopathic hypoparathyroidism (IHP) and pseudohypoparathyroidism type Ib (PHP1B). In contrast to PHP1B, which is caused by resistance toward parathyroid hormone (PTH), the genetic defects leading to IHP impair production of this important regulator of mineral ion homeostasis. So far, only five PTH mutations were shown to cause IHP, each of which is located in the hormone's pre-pro leader segment and thus impair hormone secretion. In three siblings affected by IHP, we now identified a homozygous arginine-to-cysteine mutation at position 25 (R25C) of the mature PTH(1-84) polypeptide; heterozygous family members are healthy. Depending on the assay used for evaluating these patients, plasma PTH levels were either low or profoundly elevated, thus leading to ambiguities regarding the underlying diagnosis, namely IHP or PHP1B. Consistent with increased PTH levels, recombinant [Cys25]PTH(1-84) and wild-type PTH(1-84) were secreted equally well by transfected COS-7 cells. However, synthetic [Cys25]PTH(1-34) was found to have a lower binding affinity for the PTH receptor type-1 (PTH1R) than PTH(1-34) and consequently a lower efficiency for stimulating cAMP formation in cells expressing this receptor. Consistent with these in vitro findings, long-term infusion of [Cys25]PTH(1-34) resulted only in minimal calcemic and phosphaturic responses, despite readily detectable levels of [Cys25]PTH(1-34) in plasma. The mineral ion abnormalities observed in the three IHP patients are thus most likely caused by the inherited homozygous missense PTH mutation, which reduces bioactivity of the secreted hormone. Based on these findings, screening for PTH(1-84) mutations should be considered when clinical and laboratory findings are consistent with PHP1B, but GNAS methylation changes have been excluded. Differentiating between IHP and PHP1B has considerable implications for genetic counseling, therapy, and long-term outcome because treatment of IHP patients with inappropriately high doses of active vitamin D and calcium can contribute to development of nephrocalcinosis and chronic kidney disease. © 2015 American Society for Bone and Mineral Research. Mon, 08 Jun 2015 00:31:50 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2517 Autosomal recessive osteopetrosis (ARO) is a rare genetic bone disease with genotypic and phenotypic heterogeneity, sometimes translating into delayed diagnosis and treatment. In particular, cases of intermediate severity often constitute a diagnostic challenge and represent good candidates for exome sequencing. Here, we describe the tortuous path to identification of the molecular defect in two siblings, in which osteopetrosis diagnosed in early childhood followed a milder course, allowing them to reach the adult age in relatively good conditions with no specific therapy. No clearly pathogenic mutation was identified either with standard amplification and resequencing protocols or with exome sequencing analysis. While evaluating the possible impact of a 3'UTR variant on the TCIRG1 expression, we found a novel single nucleotide change buried in the middle of intron 15 of the TCIRG1 gene, about 150 nucleotides away from the closest canonical splice site. By sequencing a number of independent cDNA clones covering exons 14 to 17, we demonstrated that this mutation reduced splicing efficiency but did not completely abrogate the production of the normal transcript. Prompted by this finding, we sequenced the same genomic region in 33 patients from our unresolved ARO cohort and found three additional novel single nucleotide changes in a similar location and with a predicted disruptive effect on splicing, further confirmed in one of them at the transcript level. Overall, we identified an intronic region in TCIRG1 that seems to be particularly prone to splicing mutations, allowing the production of a small amount of protein sufficient to reduce the severity of the phenotype usually associated with TCIRG1 defects. On this basis, we would recommend including TCIRG1 not only in the molecular work-up of severe infantile osteopetrosis but also in intermediate cases and carefully evaluating the possible effects of intronic changes. © 2015 American Society for Bone and Mineral Research. Thu, 21 May 2015 05:05:21 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2514 Certain missense mutations affecting LRP5 cause high bone mass (HBM) in humans. Based on in vitro evidence, HBM LRP5 receptors are thought to exert their effects by providing resistance to binding/inhibition of secreted LRP5 inhibitors such as sclerostin (SOST) and Dickkopf homolog-1 (DKK1). We previously reported the creation of two Lrp5 HBM knock-in mouse models, in which the human p.A214V or p.G171V missense mutations were knocked into the endogenous Lrp5 locus. To determine whether HBM knock-in mice are resistant to SOST- or DKK1-induced osteopenia, we bred Lrp5 HBM mice with transgenic mice that overexpress human SOST in osteocytes (8kbDmp1-SOST) or mouse DKK1 in osteoblasts and osteocytes (2.3kbCol1a1-Dkk1). We observed that the 8kbDmp1-SOST transgene significantly lowered whole-body bone mineral density (BMD), bone mineral content (BMC), femoral and vertebral trabecular bone volume fraction (BV/TV), and periosteal bone-formation rate (BFR) in wild-type mice but not in mice with Lrp5 p.G171V and p.A214V alleles. The 2.3kbCol1a1-Dkk1 transgene significantly lowered whole-body BMD, BMC, and vertebral BV/TV in wild-type mice and affected p.A214V mice more than p.G171V mice. These in vivo data support in vitro studies regarding the mechanism of HBM-causing mutations, and imply that HBM LRP5 receptors differ in their relative sensitivity to inhibition by SOST and DKK1. © 2015 American Society for Bone and Mineral Research. Tue, 22 Sep 2015 04:17:55 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2527 Myocardial infarction (MI) is a major cause of death worldwide. Epidemiological studies have linked vitamin D deficiency to MI incidence. Because fibroblast growth factor-23 (FGF23) is a master regulator of vitamin D hormone production and has been shown to be associated with cardiac hypertrophy per se, we explored the hypothesis that FGF23 may be a previously unrecognized pathophysiological factor causally linked to progression of cardiac dysfunction post-MI. Here, we show that circulating intact Fgf23 was profoundly elevated, whereas serum vitamin D hormone levels were suppressed, after induction of experimental MI in rat and mouse models, independent of changes in serum soluble Klotho or serum parathyroid hormone. Both skeletal and cardiac expression of Fgf23 was increased after MI. Although the molecular link between the cardiac lesion and circulating Fgf23 concentrations remains to be identified, our study has uncovered a novel heart–bone–kidney axis that may have important clinical implications and may inaugurate the new field of cardio-osteology. © 2015 American Society for Bone and Mineral Research. Wed, 06 May 2015 10:05:28 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2538 Although nullizygous loss of NF1 leads to myeloid malignancies, haploinsufficient loss of NF1 (Nf1) has been shown to contribute to osteopenia and osteoporosis which occurs in approximately 50% of neurofibromatosis type 1 (NF1) patients. Bone marrow mononuclear cells of haploinsufficient NF1 patients and Nf1+/– mice exhibit increased osteoclastogenesis and accelerated bone turnover; however, the culprit hematopoietic lineages responsible for perpetuating these osteolytic manifestations have yet to be elucidated. Here we demonstrate that conditional inactivation of a single Nf1 allele within the myeloid progenitor cell population (Nf1-LysM) is necessary and sufficient to promote multiple osteoclast gains-in-function, resulting in enhanced osteoclastogenesis and accelerated osteoclast bone lytic activity in response to proresorptive challenge in vivo. Surprisingly, mice conditionally Nf1 heterozygous in mature, terminally differentiated osteoclasts (Nf1-Ctsk) do not exhibit any of these skeletal phenotypes, indicating a critical requirement for Nf1 haploinsufficiency at a more primitive/progenitor stage of myeloid development in perpetuating osteolytic activity. We further identified p21Ras-dependent hyperphosphorylation of Pu.1 within the nucleus of Nf1 haploinsufficient myelomonocytic osteoclast precursors, providing a novel therapeutic target for the potential treatment of NF1 associated osteolytic manifestations. © 2015 American Society for Bone and Mineral Research Thu, 21 May 2015 05:05:05 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2533 Increased diacylglycerol (DAG) levels are observed in numerous pathologies, including conditions associated with bone loss. However, the effects of DAG accumulation on the skeleton have never been directly examined. Because DAG is strictly controlled by tissue-specific diacylglycerol kinases (DGKs), we sought to examine the biological consequences of DAG accumulation on bone homeostasis by genetic deletion of DGKζ, a highly expressed DGK isoform in osteoclasts (OCs). Strikingly, DGKζ-/- mice are osteoporotic because of a marked increase in OC numbers. In vitro, DGKζ-/- bone marrow macrophages (BMMs) form more numerous, larger, and highly resorptive OCs. Surprisingly, although increased DAG levels do not alter receptor activator of NF-κB (RANK)/RANK ligand (RANKL) osteoclastogenic pathway, DGKζ deficiency increases responsiveness to the proliferative and pro-survival cytokine macrophage colony-stimulating factor (M-CSF). We find that M-CSF is responsible for increased DGKζ-/- OC differentiation by promoting higher expression of the transcription factor c-Fos, and c-Fos knockdown in DGKζ-/- cultures dose-dependently reduces OC differentiation. Using a c-Fos luciferase reporter assay lacking the TRE responsive element, we also demonstrate that M-CSF induces optimal c-Fos expression through DAG production. Finally, to demonstrate the importance of the M-CSF/DGKζ/DAG axis on regulation of c-Fos during osteoclastogenesis, we turned to PLCγ2+/- BMMs, which have reduced DAG levels and form fewer OCs because of impaired expression of the master regulator of osteoclastogenesis NFATc1 and c-Fos. Strikingly, genetic deletion of DGKζ in PLCγ2+/- mice rescues OC formation and normalizes c-Fos levels without altering NFATc1 expression. To our knowledge, this is the first report implicating M-CSF/DGKζ/DAG axis as a critical regulator of bone homeostasis via its actions on OC differentiation and c-Fos expression. © 2015 American Society for Bone and Mineral Research. Wed, 26 Aug 2015 08:07:34 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2528 Physical activity is essential to maintain skeletal mass and structure, but its effect seems to diminish with age. To test the hypothesis that bone becomes less sensitive to mechanical strain with age, we used a combined in vivo/in silico approach. We investigated how maturation and aging influence the mechanical regulation of bone formation and resorption to 2 weeks of noninvasive in vivo controlled loading in mice. Using 3D in vivo morphometrical assessment of longitudinal microcomputed tomography images, we quantified sites in the mouse tibia where bone was deposited or resorbed in response to controlled in vivo loading. We compared the (re)modeling events (formation/resorption/quiescent) to the mechanical strains induced at these sites (predicted using finite element analysis). Mice of all age groups (young, adult, and elderly) responded to loading with increased formation and decreased resorption, preferentially at high strains. Low strains were associated with no anabolic response in adult and elderly mice, whereas young animals showed a strong response. Adult animals showed a clear separation between strain ranges where formation and resorption occurred but without an intermediate quiescent “lazy zone”. This strain threshold disappeared in elderly mice, as mechanically induced (re)modeling became dysregulated, apparent in an inability to inhibit resorption or initiate formation. Contrary to what is generally believed until now, aging does not shift the mechanical threshold required to initiate formation or resorption, but rather blurs its specificity. These data suggest that pharmaceutical strategies augmenting physical exercise should consider this dysfunction in the mechanical regulation of bone (re)modeling to more effectively combat age-related bone loss. © 2015 American Society for Bone and Mineral Research. Fri, 01 May 2015 23:03:26 +0000 http://onlinelibrary.wiley.com/resolve/doi?DOI=10.1002%2Fjbmr.2518 Osteogenesis imperfecta (OI) is a heterogeneous heritable connective tissue disorder associated with reduced bone mineral density and skeletal fragility. Bone is inherently mechanosensitive, with bone strength being proportional to muscle mass and strength. Physically active healthy children accrue more bone than inactive children. Children with type I OI exhibit decreased exercise capacity and muscle strength compared with healthy peers. It is unknown whether this muscle weakness reflects decreased physical activity or a muscle pathology. In this study, we used heterozygous G610C OI model mice (+/G610C), which model both the genotype and phenotype of a large Amish OI kindred, to evaluate hindlimb muscle function and physical activity levels before evaluating the ability of +/G610C mice to undergo a treadmill exercise regimen. We found +/G610C mice hindlimb muscles do not exhibit compromised muscle function, and their activity levels were not reduced relative to wild-type mice. The +/G610C mice were also able to complete an 8-week treadmill regimen. Biomechanical integrity of control and exercised wild-type and +/G610C femora were analyzed by torsional loading to failure. The greatest skeletal gains in response to exercise were observed in stiffness and the shear modulus of elasticity with alterations in collagen content. Analysis of tibial cortical bone by Raman spectroscopy demonstrated similar crystallinity and mineral/matrix ratios regardless of sex, exercise, and genotype. Together, these findings demonstrate +/G610C OI mice have equivalent muscle function, activity levels, and ability to complete a weight-bearing exercise regimen as wild-type mice. The +/G610C mice exhibited increased femoral stiffness and decreased hydroxyproline with exercise, whereas other biomechanical parameters remain unaffected, suggesting a more rigorous exercise regimen or another exercise modality may be required to improve bone quality of OI mice. © 2015 American Society for Bone and Mineral Research. Thu, 14 May 2015 09:32:47 +0000