Journal of Bone and Mineral Research

Journal of Bone and Mineral Research

RANKL Inhibition in Fibrous Dysplasia of Bone: A Preclinical Study in a Mouse Model of the Human Disease

21-08-2019 – Biagio Palmisano, Emanuela Spica, Cristina Remoli, Rossella Labella, Annamaria Di Filippo, Samantha Donsante, Fabiano Bini, Domenico Raimondo, Franco Marinozzi, Alan Boyde, Pamela Robey, Alessandro Corsi, Mara Riminucci

Journal Article

ABSTRACTFibrous dysplasia of bone/McCune‐Albright syndrome (Polyostotic FD/MAS; OMIM#174800) is a crippling skeletal disease caused by gain‐of‐function mutations of Gsα. Enhanced bone resorption is a recurrent histological feature of FD and a major cause of fragility of affected bones. Previous work suggests that increased bone resorption in FD is driven by RANKL and some studies have shown that the anti‐RANKL monoclonal antibody, denosumab, reduces bone turnover and bone pain in FD patients. However, the effect of RANKL inhibition on the histopathology of FD and its impact on the natural history of the disease remain to be assessed. In this study, we treated the EF1α‐GsαR201C mice, which develop an FD‐like phenotype, with an anti‐mouse RANKL monoclonal antibody. We found that the treatment induced marked radiographic and microscopic changes at affected skeletal sites in 2‐month‐old mice. The involved skeletal segments became sclerotic due to the deposition of new, highly mineralized bone within developing FD lesions and showed a higher mechanical resistance compared to affected segments from untreated transgenic mice. Similar changes were also detected in older mice with a full‐blown skeletal phenotype. The administration of anti‐mouse RANKL antibody arrested the growth of established lesions and, in young mice, prevented the appearance of new ones. However, after drug withdrawal, the newly formed bone was remodelled into FD tissue and the disease progression resumed in young mice. Taken together, our results show that the anti‐RANKL antibody significantly affected the bone pathology and natural history of FD in the mouse. Pending further work on the prevention and management of relapse after treatment discontinuation, our preclinical study suggests that RANKL inhibition may be an effective therapeutic option for FD patients. © 2019 American Society for Bone and Mineral Research.

Hop2 Interacts with ATF4 to Promote Osteoblast Differentiation

21-08-2019 – Yang Zhang, Tonghui Lin, Na Lian, Huan Tao, Cong Li, Lingzhen Li, Xiangli Yang

Journal Article

Abstract
Activating transcription factor 4 (ATF4) is a member of the basic leucine zipper (b
Zip) transcription factor family required for the terminal differentiation of osteoblasts. Despite its critical importance as one of the three main osteoblast differentiation transcription factors, regulators of osteoblast terminal maturation remain poorly defined. Here we report the identification of homologous pairing protein 2 (Hop2) as a dimerization partner of ATF4 in osteoblasts via the yeast two hybrid system. Deletional mapping revealed that the Zip domain of Hop2 is necessary and sufficient to bind ATF4 and to enhance ATF4‐dependent transcription. Ectopic Hop2 expression in preosteoblasts increased endogenous ATF4 protein content and accelerates osteoblast differentiation. Mice lacking Hop2 (Hop2‐/‐) have a normal stature but exhibit an osteopenic phenotype similar to the one observed in Atf4‐/‐ mice, albeit milder, which is associated with decreased Osteocalcin m
RNA expression and reduced Type I collagen synthesis. Compound heterozygous mice (Atf4+/‐:Hop2+/‐) display identical skeletal defects to those seen in Hop2‐/‐ mice. These results indicate that Hop2 plays a previous unknown role as a determinant of osteoblast maturation via its regulation of ATF4 transcriptional activity. Our work for the first time reveals a function of Hop2 beyond its role in guiding the alignment of homologous chromosomes.
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Reply to: Change in Bone Density and Reduction in Fracture Risk: A Meta‐Regression of Published Trials

21-08-2019 – Mary L Bouxsein, Richard Eastell, Li‐Yung Liu, Lucy A Wu, Anne E Papp, Andreas Grauer, Fernando Marin, Jane A Cauley, Douglas C Bauer, Dennis M Black, for the FNIH Bone Quality Project

Letter

Genetic screening in a large Chinese cohort of childhood onset hypoparathyroidism by next‐generation sequencing combined with TBX1‐MLPA

21-08-2019 – Yabing Wang, Min Nie, Ou Wang, Yuepeng Li, Yan Jiang, Mei Li, Weibo Xia, Xiaoping Xing

Journal Article

ABSTRACTAt least 15 candidate genes have been implicated in hypoparathyroidism (HP). However, comprehensive screening of causative genes for HP is lacking. Here, we investigated the genotype spectrum in a large group of Chinese patients with childhood onset HP. A total of 173 patients with childhood onset HP were analyzed using targeted next‐generation sequencing (NGS), including 15 candidate genes combined with multiplex ligation‐dependent probe amplification (MLPA) of the TBX1 gene. Twenty‐seven pathogenic or likely pathogenic mutations in five genes (TBX1, AIRE, GATA3, FAM111A, and CASR) including 14 novel variants in 23 patients, and 12 variants of uncertain clinical significance in five genes (GATA3, CASR, FAM111A, GCM2, and PTH) in 11 patients, were identified by NGS. And an entire gene deletion of TBX1 in 25 patients was found by TBX1‐MLPA. Combined with clinical data, 26 (15.0%) cases of Di
George syndrome (OMIM #188400), nine (5.2%) autoimmune polyglandular syndrome type 1 (OMIM #240300), eight (4.6%) autosomal dominant hypocalcemia type 1 (OMIM #601198), four (2.3%) hypoparathyroidism‐deafness‐renal dysplasia syndrome (OMIM #146255), and one (0.6%) Kenny‐Caffey syndrome type 2 (OMIM #127000) were verified. Among them, 16/26 (61.5%) of Di
George syndrome cases were undiagnosed due to the lack of obvious clinical clues before genetic testing. The onset age of patients with mutations (2.8 0.1, 9.6 years) was significantly earlier than those without mutations (13.0 8.8, 15.0) (P < 0.001). Family history, early onset age especially prior to 5 years old, and extra‐parathyroid manifestations were clues for hereditary HP. The combined targeted NGS and TBX‐1 MLPA were conveniently and effectively used for comprehensive genetic screening in this large Chinese cohort of childhood onset HP. Genetic defects were identified in 27.7% of early‐onset HP patients, including four kinds of syndromic HP and one isolated HP. A total of 14 novel mutations were detected, which expands the mutation spectrum of hypoparathyroidism.
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Zoledronate for the Prevention of Bone Loss in Women Discontinuing Denosumab Treatment. A Prospective 2‐year Clinical Trial

21-08-2019 – Athanasios D. Anastasilakis, Socrates E. Papapoulos, Stergios A. Polyzos, Natasha M. Appelman‐Dijkstra, Polyzois Makras

Journal Article

ABSTRACTCessation of denosumab treatment is associated with increases in bone turnover above baseline values and rapid bone loss. We investigated the efficacy of zoledronate to prevent this bone loss in women with postmenopausal osteoporosis who were treated with denosumab (mean duration 2.2 years) and discontinued treatment after achieving osteopenia. Women were randomized to receive a single zoledronate 5 mg infusion (n = 27) or two additional denosumab 60 mg injections (n = 30). Both groups were followed for a total period of 24 months. At 24 months LS‐BMD was not different from baseline in the ZOL group, but decreased in the Dmab group by 4.82 ± 0.7% (p < 0.001) from the 12‐month value; the difference in BMD changes between the two groups, the primary endpoint of the study, was statistically significant (p = 0.025). Results of FN‐BMD changes were similar. Zoledronate infusion was followed by small but significant increases in serum Procollagen type 1 N‐terminal propeptide (P1NP) and C‐terminal telopeptide of type 1 collagen (CTX) during the first year and stabilization thereafter. In the denosumab group, bone turnover marker values did not change during the first 12 months but increased significantly at 15 months and in the majority of women these remained elevated at 24 months. Neither baseline nor 12‐month bone turnover marker values were associated with BMD changes in either group of women. In the denosumab group, 3 patients sustained vertebral fractures (2 patients multiple clinical, 1 patient morphometric) while 1 patient in the zoledronate group sustained clinical vertebral fractures 12 months after the infusion. In conclusion, a single intravenous infusion of zoledronate given 6 months after the last Dmab injection prevents bone loss for at least two years independently of the rate of bone turnover. Follow‐up is recommended, as in a few patients ZOL treatment might not have the expected effect at 2 years.
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Change in Bone Density and Reduction in Fracture Risk: A Meta‐Regression of Published Trials

21-08-2019 – Pierre JM Bergmann

Letter

Pain at Multiple Sites Is Associated With Prevalent and Incident Fractures in Older Adults

19-08-2019 – Feng Pan, Jing Tian, Dawn Aitken, Flavia Cicuttini, Graeme Jones

Journal Article

ABSTRACTMusculoskeletal pain is common and typically occurs at multiple sites. Pain has been shown to be associated with falls risk; however, whether an increased risk for falls associated with multisite pain (MSP) translates into an increased risk of fractures has not been investigated. This study aimed to examine the association of number of painful sites with prevalent and incident fractures. Data from a longitudinal population‐based study of older adults (mean age 63 years) were utilized. Follow‐up was performed at 2.6, 5.1, and 10.7 years later, respectively. Presence/absence of pain at the neck, back, hands, shoulders, hips, knees, and feet was assessed by questionnaire at baseline. Participants were classified into three groups according to the total number of painful sites: zero to two, three to four, and five to seven. Fractures were self‐reported at each time point. BMD was measured by DXA. Falls risk was calculated based on the Short‐Form Physiological Profile Assessment. Log‐binomial regression was used for the analyses. There were 450 fractures at baseline and 154 new fractures reported during a mean follow‐up period of 10.7 years (range 9.2 to 12.5 years). In multivariable analyses, number of painful sites was associated with prevalent fractures at any and nonvertebral site. Furthermore, participants with five to seven painful sites had an increased risk of incident fractures at any site (RR 1.69; 95% CI, 1.13 to 2.53); major site, including the femur, radius, ulnar, vertebral, rib, and humerus (RR 2.17; 95% CI 1.12 to 4.22); and vertebral site (RR 6.44, 95% CI, 1.64 to 25.33) compared with those with pain at zero to two sites. These associations remained statistically significant after further adjustment for falls risk and BMD. Pain at multiple sites was associated with incident fracture risk in a dose‐response manner, suggesting that widespread pain is an independent contributor to fracture risk. The potential for pain management in fracture prevention warrants further exploration. © 2019 American Society for Bone and Mineral Research.

IRS‐1 Functions as a Molecular Scaffold to Coordinate IGF‐I/IGFBP‐2 Signaling During Osteoblast Differentiation

19-08-2019 – Gang Xi, Xinchun Shen, Clifford J Rosen, David R Clemmons

Gender‐Affirming Hormone Treatment Decreases Bone Turnover in Transwomen and Older Transmen

19-08-2019 – Mariska C Vlot, Chantal M Wiepjes, Renate T Jongh, Guy T’Sjoen, Annemieke C Heijboer, Martin den Heijer

Journal Article

ABSTRACTSex steroids play a key role in bone turnover and preserving BMD; hence, gender‐affirming hormone treatment (HT) in transgender people affects bone metabolism. Most studies have looked into the effect of HT on changes in BMD; however, they do not provide insights into changes in bone metabolism caused by HT. This study investigated changes in bone turnover markers (BTMs) and sclerostin, as well as their correlations with change in BMD in transwomen and transmen during the first year of HT. Transwomen received estradiol and antiandrogens; transmen received testosterone. Sclerostin; P1NP; alkaline phosphatase (ALP); CTx; and BMD of the total hip, the femoral neck, and the lumbar spine were evaluated at baseline and after 1 year of HT. There were 121 transwomen (median age 30 years, interquartile range IQR 24 to 41 years) and 132 transmen (median age 24 years, IQR 21 to 33 years) included in the study. In transwomen, ALP decreased in 19% (95% CI, –21 to–16), CTx in 11% (95% CI, –18 to–4), and sclerostin in 8% (95%CI, –13 to–4) of study participants after 1 year of HT. In contrast, in transmen P1NP, ALP, and sclerostin increased in 33% (95% CI, 24 to 42), 16% (95% CI, 12 to 20), and 15% (95% CI, 10 to 20) of study participants, respectively, after 1 year of HT. No age differences were seen in transwomen, whereas in transmen aged ≥50 years a decrease in all BTMs was found in contrast with the other age groups. These transmen had low estrogen concentration at the start of HT based on their postmenopausal state before the start of HT; their estradiol concentrations increased during testosterone treatment. Changes in BTMs and BMD were weakly correlated (correlation coefficient all <0.30). To conclude, 1 year of HT resulted in decreased bone turnover in transwomen and older transmen, whereas it increased in younger transmen. The decrease in bone resorption in older transmen shows the importance of estrogen as a key regulator of bone turnover. © 2019 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc.

Elevated BMP and Mechanical Signaling Through YAP1/RhoA Poises FOP Mesenchymal Progenitors for Osteogenesis

19-08-2019 – Alexandra Stanley, Su‐jin Heo, Robert L Mauck, Foteini Mourkioti, Eileen M Shore

Journal Article

ABSTRACTFibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by the formation of extraskeletal bone, or heterotopic ossification (HO), in soft connective tissues such as skeletal muscle. All familial and sporadic cases with a classic clinical presentation of FOP carry a gain‐of‐function mutation (R206H; c.617 G > A) in ACVR1, a cell surface receptor that mediates bone morphogenetic protein (BMP) signaling. The BMP signaling pathway is recognized for its chondro/osteogenic‐induction potential, and HO in FOP patients forms ectopic but qualitatively normal endochondral bone tissue through misdirected cell fate decisions by tissue‐resident mesenchymal stem cells. In addition to biochemical ligand‐receptor signaling, mechanical cues from the physical environment are transduced to activate intracellular signaling, a process known as mechanotransduction, and can influence cell fates. Utilizing an established mesenchymal stem cell model of mouse embryonic fibroblasts (MEFs) from the Acvr1 R206H/+ mouse model that mimics the human disease, we demonstrated that activation of the mechanotransductive effectors Rho/ROCK and YAP1 are increased in Acvr1 R206H/+ cells. We show that on softer substrates, a condition associated with low mechanical signaling, the morphology of Acvr1 R206H/+ cells is similar to the morphology of control Acvr1 +/+ cells on stiffer substrates, a condition that activates mechanotransduction. We further determined that Acvr1 R206H/+ cells are poised for osteogenic differentiation, expressing increased levels of chondro/osteogenic markers compared with Acvr1 +/+ cells. We also identified increased YAP1 nuclear localization in Acvr1 R206H/+ cells, which can be rescued by either BMP inhibition or Rho antagonism. Our results establish Rho
A and YAP1 signaling as modulators of mechanotransduction in FOP and suggest that aberrant mechanical signals, combined with and as a result of the increased BMP pathway signaling through mutant ACVR1, lead to misinterpretation of the cellular microenvironment and a heightened sensitivity to mechanical stimuli that promotes commitment of Acvr1 R206H/+ progenitor cells to chondro/osteogenic lineages.

Clinical performance of the updated trabecular bone score (TBS) algorithm which accounts for the soft tissue thickness: The OsteoLaus Study

16-08-2019 – Enisa Shevroja, Bérengère Aubry‐Rozier, Gabriel Hans, Elena Gonzalez Rodriguez, Delphine Stoll, Olivier Lamy, Didier Hans

Journal Article

ABSTRACTRegional soft tissue may have a noise effect on trabecular bone score (TBS) and eventually alter its estimate. The current TBS software (TBS i
Nsight®) is based on an algorithm accounting for body mass index (BMI) (TBSv3.03). We aimed to explore the updated TBS algorithm that accounts for soft tissue thickness (TBSv4.0). This study was embedded in the Osteo
Laus cohort of women in Lausanne, Switzerland. Hip and lumbar spine (LS) dual‐energy X‐ray absorptiometry (DXA) scans were performed using Discovery A System (Hologic, 123, MA, USA). The incident major osteoporotic fractures (MOFs) were assessed from vertebral fracture assessments using the Genant`s method or questionnaires (non‐vertebral MOF). We assessed the correlations of bone mineral density (BMD) or TBS with body composition parameters; MOF prediction ability of both versions of TBS; and the differences between FRAX adjusted for TBSv3.03 or TBSv4.0. In total, 1,362 women with mean age (SD) 64.4 (7.5) years and mean BMI (SD) 25.9 (4.5) kg/m2 were followed up for 4.4 years and 132 experienced a MOF. All the anthropometric measurements of our interest were positively correlated with LS, femoral neck or hip BMD and TBSv4.0; whereas with TBSv3.03 their correlations were negative. In the models adjusted for age, soft tissue thickness, osteoporotic treatment and LS‐BMD, for each SD decline in TBSv3.03, there was a 43% (OR (95% CI): 1.43 (1.12‐1.83)) increase in the odds of having MOF; whereas for each SD decline in TBSv4.0, there was a 54% (OR (95% CI): 1.54 (1.18‐2.00)) increase in the odds of having a MOF. Both FRAXs were very strongly correlated and the mild differences were present in the already highly risked women for MOF. This study demonstrates that TBSv4.0 overcomes the debatable residual negative correlation of the current TBS with body size and composition parameters, postulating itself as free from the previously acknowledged technical limitation of TBS.
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Post‐fracture Risk Assessment: Target Central Sited Fractures First! A Substudy of NoFRACT

16-08-2019 – Tove T Borgen, åshild Bjørnerem, Lene B Solberg, Camilla Andreasen, Cathrine Brunborg, May‐Britt Stenbro, Lars M Hübschle, Anne Froholdt, Wender Figved, Ellen M Apalset, Jan‐Erik Gjertsen, Trude Basso, Ida Lund, Ann K Hansen, Jens‐Meinhard Stutzer, Tone K Omsland, Lars Nordsletten, Frede Frihagen, Erik F Eriksen

Journal Article

Fractures at central sites are associated with lower BMD at the femoral neck, total hip, and the site with lowest T‐score, lower TBS, and higher prevalence of vertebral fractures. BMD = bone mineral density; TBS = trabecular bone score.
ABSTRACTThe location of osteoporotic fragility fractures adds crucial information to post‐fracture risk estimation. Triaging patients according to fracture site for secondary fracture prevention can therefore be of interest to prioritize patients considering the high imminent fracture risk. The objectives of this cross‐sectional study were therefore to explore potential differences between central (vertebral, hip, proximal humerus, pelvis) and peripheral (forearm, ankle, other) fractures. This substudy of the Norwegian Capture the Fracture Initiative (No
FRACT) included 495 women and 119 men ≥50 years with fragility fractures. They had bone mineral density (BMD) of the femoral neck, total hip, and lumbar spine assessed using dual‐energy X‐ray absorptiometry (DXA), trabecular bone score (TBS) calculated, concomitantly vertebral fracture assessment (VFA) with semiquantitative grading of vertebral fractures (SQ1–SQ3), and a questionnaire concerning risk factors for fractures was answered. Patients with central fractures exhibited lower BMD of the femoral neck (765 versus 827 mg/cm2), total hip (800 versus 876 mg/cm2), and lumbar spine (1024 versus 1062 mg/cm2); lower mean TBS (1.24 versus 1.28); and a higher proportion of SQ1‐SQ3 fractures (52.0% versus 27.7%), SQ2–SQ3 fractures (36.8% versus 13.4%), and SQ3 fractures (21.5% versus 2.2%) than patients with peripheral fractures (all p < 0.05). All analyses were adjusted for sex, age, and body mass index (BMI); and the analyses of TBS and SQ1–SQ3 fracture prevalence was additionally adjusted for BMD). In conclusion, patients with central fragility fractures revealed lower femoral neck BMD, lower TBS, and higher prevalence of vertebral fractures on VFA than the patients with peripheral fractures. This suggests that patients with central fragility fractures exhibit more severe deterioration of bone structure, translating into a higher risk of subsequent fragility fractures and therefore they should get the highest priority in secondary fracture prevention, although attention to peripheral fractures should still not be diminished. © 2019 American Society for Bone and Mineral Research. © 2019 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Standard vs Cyclic Teriparatide and Denosumab Treatment for Osteoporosis: A Randomized Trial

16-08-2019 – Felicia Cosman, Donald McMahon, David Dempster, Jeri W. Nieves

Journal Article

Abstract
In the absence of an intervening antiresorptive agent, cyclic administration of teriparatide does not increase BMD more than standard daily therapy. Since denosumab is a potent antiresorptive agent with a rapid off‐effect, we hypothesized that it might be the optimal agent to help maximize bone gains with cyclic teriparatide. In this 3 year protocol, 70 postmenopausal women with osteoporosis were randomized to: 18 months teriparatide followed by 18 months denosumab (Standard) or 3 separate 12 month cycles of 6 months teriparatide followed by 6 months denosumab (Cyclic). BMD (DXA) measurements of lumbar spine (LS), total hip (TH), femoral neck (FN), and 1/3 radius (RAD) were performed every 6 months and Total Body Bone Mineral (TBBM) at 18 and 36 months. Baseline descriptive characteristics did not differ between groups except for a minimal difference in LS BMD, but not T‐Score (mean age 65, mean LS T‐Score ‐2.7). In the Standard group, BMD increments at 36 months were: LS 16%, TH 4%, FN 3% and TBBM 4.8% (all p<0.001 vs baseline). In the Cyclic group, 36‐month BMD increments were similar: LS 12%, TH 4%, FN 4%, TBBM 4.1% (all p<0.001 vs baseline). At 36 months, the LS BMD increase with Standard was slightly larger than with Cyclic (p=0.04), but at 18 months, in the Cyclic group, there was no decline in RAD or TBBM (p=0.007 and <0.001, respectively vs Standard). Although the Cyclic regimen did not improve BMD compared with Standard at 36 months, there appeared to be a benefit at 18 months, especially in the highly cortical skeletal sites. This could be clinically relevant in patients at high imminent risk of fracture, particularly at nonvertebral sites.
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Adding Marrow Adiposity and Cortical Porosity to Femoral Neck Areal Bone Mineral Density Improves the Discrimination of Women With Nonvertebral Fractures From Controls

15-08-2019 – Roger Zebaze, Marit Osima, Minh Bui, Marko Lukic, Xiaofang Wang, Ali Ghasem‐Zadeh, Erik F Eriksen, Angela Vais, Catherine Shore‐Lorenti, Peter R Ebeling, Ego Seeman, Åshild Bjørnerem

Journal Article

ABSTRACTAdvancing age is accompanied by a reduction in bone formation and remodeling imbalance, which produces microstructural deterioration. This may be partly caused by a diversion of mesenchymal cells towards adipocytes rather than osteoblast lineage cells. We hypothesized that microstructural deterioration would be associated with an increased marrow adiposity, and each of these traits would be independently associated with nonvertebral fractures and improve discrimination of women with fractures from controls over that achieved by femoral neck (FN) areal bone mineral density (a
BMD) alone. The marrow adiposity and bone microstructure were quantified from HR‐p
QCT images of the distal tibia and distal radius in 77 women aged 40 to 70 years with a recent nonvertebral fracture and 226 controls in Melbourne, Australia. Marrow fat measurement from HR‐p
QCT images was validated using direct histologic measurement as the gold standard, at the distal radius of 15 sheep, with an agreement (R2 = 0.86, p < 0.0001). Each SD higher distal tibia marrow adiposity was associated with 0.33 SD higher cortical porosity, and 0.60 SD fewer, 0.24 SD thinner, and 0.72 SD more‐separated trabeculae (all p < 0.05). Adjusted for age and FN a
BMD, odds ratios (ORs) (95% CI) for fracture per SD higher marrow adiposity and cortical porosity were OR, 3.39 (95% CI, 2.14 to 5.38) and OR, 1.79 (95% CI, 1.14 to 2.80), respectively. Discrimination of women with fracture from controls improved when cortical porosity was added to FN a
BMD and age (area under the receiver‐operating characteristic curve AUC 0.778 versus 0.751, p = 0.006) or marrow adiposity was added to FN a
BMD and age (AUC 0.825 versus 0.751, p = 0.002). The model including FN a
BMD, age, cortical porosity, trabecular thickness, and marrow adiposity had an AUC = 0.888. Results were similar for the distal radius. Whether marrow adiposity and cortical porosity indices improve the identification of women at risk for fractures requires validation in prospective studies. © 2019 American Society for Bone and Mineral Research.

A New Understanding of the Role of IL‐1 in Age‐Related Intervertebral Disc Degeneration in a Murine Model

15-08-2019 – Deborah J Gorth, Irving M Shapiro, Makarand V Risbud

Journal Article

ABSTRACTIncreased cytokine expression, in particular interleukin‐1β (IL‐1β), is considered a hallmark of intervertebral disc degeneration. However, the causative relationship between IL‐1 and age‐dependent degeneration has not been established. To investigate the role of IL‐1 in driving age‐related disc degeneration, we studied the spine phenotype of global IL‐1α/β double knockout (IL‐1KO) mice at 12 and 20 months. Multiplex ELISA analysis of blood revealed significant reductions in the concentrations of IFN‐γ, IL‐5, IL‐15, TNF‐α, IP‐10, and a trend of reduced concentrations of IL‐10, macrophage inflammatory protein 1α (MIP‐1α), keratinocyte chemoattractant/human growth‐regulated oncogene (KC/GRO), and IL‐6. However, the circulating level of MIP‐2, a neutrophil chemoattractant, was increased in the IL‐1KO. The alterations in systemic cytokine levels coincided with altered bone morphology—IL‐1KO mice exhibited significantly thicker caudal cortical bone at 12 and 20 months. Despite these systemic inflammatory and bony changes, IL‐1 deletion only minimally affected disc health. Both wild‐type (WT) and IL‐1KO mice showed age‐dependent disc degeneration. Unexpectedly, rather than protecting the animals from degeneration, the aging phenotype was more pronounced in IL‐1KO animals: knockout mice evidenced significantly more degenerative changes in the annulus fibrosis (AF) together with alterations in collagen type and maturity. At 20 months, there were no changes in nucleus pulposus (NP) extracellular matrix composition or cellular marker expression; however, the IL‐1KO NP cells occupied a smaller proportion of the NP compartment that those of WT controls. Taken together, these results show that IL‐1 deletion altered the systemic inflammatory environment and vertebral bone morphology. However, instead of protecting discs from age‐related disc degeneration, global IL‐1 deletion amplified the degenerative phenotype. © 2019 American Society for Bone and Mineral Research.

Klf4 Promotes Dentinogenesis and Odontoblastic Differentiation via Modulation of TGF‐β Signaling Pathway and Interaction With Histone Acetylation

15-08-2019 – Huangheng Tao, Heng Lin, Zheyi Sun, Fei Pei, Jie Zhang, Shuo Chen, Huan Liu, Zhi Chen

Journal Article

ABSTRACTTranscription factors bind to cell‐specific cis‐regulatory elements, such as enhancers and promoters, to initiate much of the gene expression program of different biological process. Odontoblast differentiation is a necessary step for tooth formation and is also governed by a complex gene regulatory network. Our previous in vitro experiments showed that Krüppel‐like factor 4 (KLF4) can promote odontoblastic differentiation of both mouse dental papillary cells (m
DPCs) and human dental pulp cells; however, its mechanism remains unclear. We first used Wnt1‐Cre; KLF4fx/fx (Klf4 c
KO) mice to examine the role of KLF4 during odontoblast differentiation in vivo and demonstrated significantly impaired dentin mineralization and enlarged pulp/root canals. Additionally, combinatory analysis using RNA‐seq and ATAC‐seq revealed genomewide direct regulatory targets of KLF4 in mouse odontoblasts. We found that KLF4 can directly activate the TGF‐β signaling pathway at the beginning of odontoblast differentiation with Runx2 as a cofactor. Furthermore, we found that KLF4 can directly upregulate the expression levels of Dmp1 and Sp7, which are markers of odontoblastic differentiation, through binding to their promoters. Interestingly, as a transcription factor, KLF4 can also recruit histone acetylase as a regulatory companion to the downstream target genes to positively or negatively regulate transcription. To further investigate other regulatory companions of KLF4, we chose histone acetylase HDAC3 and P300. Immunoprecipitation demonstrated that KLF4 interacted with P300 and HDAC3. Next, Ch
IP analysis detected P300 and HDAC3 enrichment on the promoter region of KLF4 target genes Dmp1 and Sp7. HDAC3 mainly interacted with KLF4 on day 0 of odontoblastic induction, whereas P300 interacted on day 7 of induction. These temporal‐specific interactions regulated Dmp1 and Sp7 transcription, thus regulating dentinogenesis. Taken together, these results demonstrated that KLF4 regulates Dmp1 and Sp7 transcription via the modulation of histone acetylation and is vital to dentinogenesis. © 2019 American Society for Bone and Mineral Research.

Growth Hormone Increases Bone Toughness and Decreases Muscle Inflammation in Glucocorticoid‐Treated Mdx Mice, Model of Duchenne Muscular Dystrophy

15-08-2019 – Sung‐Hee Yoon, Marc D Grynpas, Jane Mitchell

Journal Article

ABSTRACTThe absence of functional dystrophin with mutations of the dystrophin‐encoding gene in Duchenne muscular dystrophy (DMD) results in muscle inflammation and degeneration, as well as bone fragility. Long‐term glucocorticoid therapy delays the muscular disease progression but suppresses growth hormone secretion, resulting in short stature and further deleterious effects on bone strength. This study evaluated the therapeutic potential of daily growth hormone therapy in growing mdx mice as a model of DMD. Growth hormone treatment on its own or in combination with glucocorticoids significantly improved muscle histology and function and decreased markers of inflammation in mdx mice. Glucocorticoid treatment thinned cortical bone and decreased bone strength and toughness. Despite the minimal effects of growth hormone on bone microarchitecture, it significantly improved biomechanical properties of femurs and vertebrae, even in the presence of glucocorticoid treatment. Together these studies suggest that the use of growth hormone in DMD should be considered for improvements to muscle and bone health. © 2019 American Society for Bone and Mineral Research.

Undercarboxylated Osteocalcin Improves Insulin‐Stimulated Glucose Uptake in Muscles of Corticosterone‐Treated Mice

15-08-2019 – Xuzhu Lin, Lewan Parker, Emma McLennan, Alan Hayes, Glenn McConell, Tara C Brennan‐Speranza, Itamar Levinger

Journal Article

ABSTRACTShort‐term administration of glucocorticoids (GCs) impairs muscle insulin sensitivity at least in part via the reduction of undercarboxylated osteocalcin (uc
OC). However, whether uc
OC treatment reverses the GC‐induced muscle insulin resistance remains unclear. To test the hypothesis that uc
OC directly ameliorates impaired insulin‐stimulated glucose uptake (ISGU) induced by short‐term GC administration in mice muscle and to identify the molecular mechanisms, mice were implanted with placebo or corticosterone (CS) slow‐release pellets. Two days post‐surgery, insulin‐tolerance tests (ITTs) were performed. On day 3, serum was collected and extensor digitorum longus (EDL) and soleus muscles were isolated and treated ex vivo with vehicle, uc
OC (30 ng/m
L), insulin (60 µU/m
L), or both. Circulating hormone levels, muscle glucose uptake, and muscle signaling proteins were assessed. CS administration reduced both serum osteocalcin and uc
OC levels, whole‐body insulin sensitivity, and muscle ISGU in EDL. Ex vivo uc
OC treatment restored ISGU in CS‐affected muscle, without increasing non‐insulin‐stimulated glucose uptake. In CS‐affected EDL muscle, uc
OC enhanced insulin action on phosphorylated (p‐)protein kinase B (Akt)Ser473and the p‐extracellular signal‐regulated kinase isoform 2 (ERK2)Thr202/Tyr204/total (t)ERK2 ratio, which correlated with ISGU. In CS‐affected soleus muscle, uc
OC enhanced insulin action on p‐mammalian target of rapamycin (m
TOR)Ser2481, the p‐m
TORSer2481/tm
TOR ratio, p‐Akt substrate of 160k
D (AS160)Thr642, and p‐protein kinase C (PKC) (pan)Thr410, which correlated with ISGU. Furthermore, p‐PKC (pan)Thr410 correlated with p‐Akt
Ser473 and p‐AS160Thr642. uc
OC exerts direct insulin‐sensitizing effects on CS‐affected mouse muscle, likely through an enhancement in activity of key proteins involved in both insulin and uc
OC signaling pathways. Furthermore, these effects are muscle type‐dependent. © 2019 American Society for Bone and Mineral Research.

Performance of FRAX in Women with Breast Cancer Initiating Aromatase Inhibitor Therapy: A Registry‐Based Cohort Study

15-08-2019 – William D Leslie, Suzanne N Morin, Lisa M Lix, Saroj Niraula, Eugene V McCloskey, Helena Johansson, Nicholas C Harvey, John A Kanis

Journal Article

ABSTRACTFRAX was developed to predict 10‐year probability of major osteoporotic fracture (MOF) and hip fracture in the general population. Aromatase inhibitors (AI) used in breast cancer induce loss in bone mineral density (BMD) and are reported to increase fracture risk. AI exposure is not a direct input to FRAX but is captured under “secondary osteoporosis”. To inform use of FRAX in women treated with AI, we used a population‐based registry for the Province of Manitoba, Canada, to identify women aged ≥40 years initiating AI for breast cancer with at least 12 months’ AI exposure (n = 1775), women with breast cancer not receiving AI (n = 1016), and women from the general population (n = 34,205). Among AI users, fracture probability estimated without BMD (AI use coded as secondary osteoporosis) significantly overestimated risk (10‐year observed/predicted ratio 0.56, 95% confidence interval CI 0.45–0.68; 10‐year hip fracture observed/predicted ratio 0.33, 95% CI 0.18–0.49). However, when BMD was included in the fracture probability, there was no significant difference between observed and predicted fracture risk. In Cox proportional hazards models, FRAX stratified risk of MOF, hip, and any fracture equally well in all subgroups (p‐interaction >0.1). When adjusted for FRAX score without BMD, with AI use coded as secondary osteoporosis, AI users were at significantly lower risk for MOF (hazard ratio HR = 0.78, 95% CI 0.64–0.95), hip fracture (HR = 0.46, 95% CI 0.29–0.73) and any fracture (HR = 0.75, 95% CI 0.63–0.89). AI use was no longer significantly associated with fractures when AI use was not entered as secondary osteoporosis in FRAX without BMD or when BMD was included in the FRAX calculation. In conclusion, FRAX scores stratify fracture risk equally well in women receiving AI therapy as in non‐users, but including secondary osteoporosis as a risk factor for AI users overestimates fracture risk. Our results call this practice into question. © 2019 American Society for Bone and Mineral Research.

Pentosidine Is Associated With Cortical Bone Geometry and Insulin Resistance in Otherwise Healthy Children

15-08-2019 – Joseph M Kindler, Emma M Laing, Weixi Liu, Joel A Dain, Richard D Lewis

Journal Article

ABSTRACTPentosidine is an advanced glycation end product (AGE) associated with fracture in adults with diabetes. AGE accumulation in bone collagen contributes to bone fragility but might also adversely influence bone turnover and, consequently, bone geometry. The relationships between AGEs and bone health have yet to be studied in children. Thus, the objective of this study was to assess relationships between pentosidine and cortical bone volumetric density, geometry, and estimated strength in children. Participants were otherwise healthy black and white boys and girls, ages 9 to 13 years, who were at sexual maturation stage 2 or 3 (N = 160). Tibia and radius cortical bone and muscle area (66% site) were assessed via p
QCT. In fasting sera, insulin, glucose, and pentosidine were measured. The Quantitative Insulin Sensitivity Check Index (QUICKI), a measure of insulin sensitivity, was calculated. While controlling for race, sex, maturation, and height, pentosidine negatively correlated with QUICKI (P < 0.05). In unadjusted analyses, pentosidine was associated with lower radius and tibia cortical volumetric bone mineral density, bone mineral content (Ct.
BMC), area (Ct.
Ar), and thickness (Ct.
Th); a larger radius endosteal circumference (Endo.
Circ); and lower tibia polar strength strain index (all P < 0.05). While controlling for race, sex, maturation, height, and muscle area, pentosidine was negatively associated with tibia Ct.
BMC, Ct.
Ar, and Ct.
Th but positively associated with Endo.
Circ (all P < 0.05). Linear regression revealed a significant interaction between pentosidine and QUICKI in relation to tibia Ct.
Th (pinteraction = 0.049), indicating that the negative relationship between pentosidine and Ct.
Th was stronger in those with lower QUICKI (ie, greater insulin resistance). This is the first study to report evidence of a potentially adverse influence of AGEs on bone strength in otherwise healthy children. This relationship was strongest in children with the greatest insulin resistance, supporting further work in youth with chronic metabolic health conditions. © 2019 American Society for Bone and Mineral Research.

Parathyroid Hormone Remodels Bone Transitional Vessels and the Leptin Receptor‐Positive Pericyte Network in Mice

15-08-2019 – Robin Caire, Bernard Roche, Tiphanie Picot, Carmen‐Mariana Aanei, Zhiguo He, Lydia Campos, Mireille Thomas, Luc Malaval, Laurence Vico, Marie‐Hélène Lafage‐Proust

Journal Article

ABSTRACTIntermittent parathyroid hormone (i
PTH) is anti‐osteoporotic and affects bone vessels. Transitional capillaries close to the bone surface, which express both endomucin (Edm) and CD31, bear leptin receptor‐expressing (Lep
R) perivascular cells that may differentiate into osteoblasts. Increased numbers of type H endothelial cells (THEC; ie, Edmhi/CD31hi cells assessed by flow cytometry, FACS) are associated with higher bone formation in young mice. We hypothesized that i
PTH administration impacts transitional vessels by expanding THECs. Four‐month‐old C57/Bl6J female mice were injected with PTH 1–84 (100 μg/kg/d) or saline (CT) for 7 or 14 days. We quantified Lep
R+, CD31+, Edm+ cells and THECs by FACS in hindlimb bone marrow, and Edm/Lep
R double immunolabelings on tibia cryosections. Additionally, we analyzed bone m
RNA expression of 87 angiogenesis‐related genes in mice treated with either intermittent or continuous PTH (i
PTH/c
PTH) or saline (CT) for 7, 14, and 28 days. i
PTH dramatically decreased the percentage of THECs by 78% and 90% at days 7 and 14, respectively, and of Lep
R+ cells at day 14 (–46%) versus CT. Immunolabeling quantification showed that the intracortical Edm+‐vessel density increased at day 14 under i
PTH. In the bone marrow, perivascular Lep
R+ cells, connected to each other via a dendrite network, were sparser under i
PTH at day 14 (–58%) versus CT. i
PTH decreased Lep
R+ cell coverage of transitional vessels only (–51%), whereas the number of Lep
R+ cells not attached to vessels increased in the endocortical area only (+ 49%). Transcriptomic analyses showed that i
PTH consistently upregulated PEDF, Collagen‐18α1, and TIMP‐1 m
RNA expression compared with CT and c
PTH. Finally, i
PTH increased immunolabeling of endostatin, a Collagen‐18 domain that can be cleaved and become antiangiogenic, in both endocortical (79%) and peritrabecular transitional microvessels at day 14. Our results show that i
PTH specifically remodels transitional vessels and suggest that it promotes Lep
R+ cell mobilization from these vessels close to the bone surface. © 2019 American Society for Bone and Mineral Research.

Interferon Gamma‐1b Does Not Increase Markers of Bone Resorption in Autosomal Dominant Osteopetrosis

15-08-2019 – Erik A Imel, Ziyue Liu, Dena Acton, Melissa Coffman, Netsanet Gebregziabher, Yan Tong, Michael J Econs

Journal Article

ABSTRACTIn autosomal dominant osteopetrosis type 2 (ADO2) CLCN7 mutations cause impaired osteoclast function. Severe consequences include skeletal fragility despite high bone mass, osteomyelitis, osteonecrosis, bone marrow failure, and severe cranial nerve impingement. There is no effective medical treatment for ADO2. We recruited subjects with ADO2 into a 14‐week, open‐label, pilot clinical trial of interferon gamma‐1b. Doses were titrated based on tolerability and if fasting serum C‐telopeptide (CTX) was <25% above baseline at week 8, targeting doses of 100 µg/m2 three times a week. The primary outcomes were change from baseline in CTX and N‐telopeptide/creatinine ratio (NTX/Cr) at week 14. Secondary outcomes included changes in urine calcium/creatinine ratio, bone formation markers and tolerability. Nine adults and three children were recruited. Severe manifestations of ADO2 included histories of fractures (100%), osteomyelitis (16.7%), vision loss (50%), and anemia (58.3%). Baseline CTX and NTX/Cr were generally low‐normal. Procollagen type I N‐terminal propeptide was elevated or in the upper‐normal range in 11 of 12 (91.6%) subjects. Elevations of aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were common. One subject withdrew due to rash. Five subjects achieved doses of 50 µg/m2 3 days a week, while six reached the full dose of 100 µg/m2 3 days a week. Only 3 of 11 (27.3%) completing subjects achieved the primary outcome of increasing CTX ≥25% above baseline at week 14. The mean ± SD change from baseline in CTX at week 14 was +2.2% ± 43.2%, p = 0.86). Likewise, there was no significant change in NTX/Cr (mean change –2.1%, p = 0.81). Interferon gamma‐1b was poorly tolerated. Most subjects had adverse events, and the Mental Health and Mental Component Scales of the SF‐36v2 health survey declined slightly (p < 0.05). Over 14 weeks, interferon gamma‐1b failed to significantly increase bone turnover markers in ADO2 and was poorly tolerated. Consequently, interferon gamma‐1b is unlikely to be effective for decreasing bone mass in ADO2. © 2019 American Society for Bone and Mineral Research.

Performance of FRAX and FRAX‐Based Treatment Thresholds in Women Aged 40 Years and Older: The Manitoba BMD Registry

15-08-2019 – Carolyn J Crandall, John T Schousboe, Suzanne N Morin, Lisa M Lix, William Leslie

Journal Article

ABSTRACTWe examined among women aged ≥40 years the performance of the Fracture Risk Assessment Tool (FRAX) and FRAX‐based osteoporosis treatment thresholds under the US National Osteoporosis Foundation (NOF) and UK National Osteoporosis Guideline Group (NOGG) guidelines. We used registry data for all women aged ≥40 years in Manitoba, Canada, with baseline bone mineral density (BMD) testing (n = 54,459). Incident major osteoporotic fracture (MOF), hip fracture, and clinical fracture were assessed from population‐based health services data (mean follow‐up 10.5 years). Age‐stratified hazard ratios (HR) were estimated from Cox regression models. We assessed the sensitivity, specificity, positive predictive value (PPV), number needed to screen (NNS), and number needed to treat (NNT) to prevent a fracture (assuming 20% relative risk reduction on treatment) for osteoporosis treatment thresholds under the NOF and NOGG guidelines. Femoral neck T‐score and FRAX (with and without BMD) predicted all fracture outcomes at all ages. There was good calibration in FRAX‐predicted versus observed 10‐year MOF and hip fracture probability. Overall sensitivity (PPV) for incident MOF was 25.7% (24.0%) for femoral neck T‐score ≤ –2.5; 20.3% (26.3%) for FRAX (with BMD)‐predicted 10‐year MOF risk ≥20% (NOF threshold); 27.3% (22.0%) for FRAX‐predicted 10‐year MOF risk ≥ age‐dependent cut‐off (NOGG threshold), 59.4% (19.0%) for the NOF treatment algorithm; and 28.5% (18.4%) for the NOGG treatment algorithm. Sensitivity for identifying incident MOF varied by age, ranging from 0.0% to 26.3% in women 40 to 49 years old and from 49.0% to 93.3% in women aged 80+ years. The gradient of risk for fracture prediction from femoral neck T‐score and FRAX (with and without BMD) as continuous measures was strong across the age spectrum. The sensitivity and PPV of the strategies based on dichotomous cut‐offs are low, especially among women aged 40 to 49 years (who have lowest incidence rates). Threshold‐based approaches should be reassessed, particularly in younger women. © 2019 American Society for Bone and Mineral Research.

Mechanical Competence and Bone Quality Develop During Skeletal Growth

15-08-2019 – Elizabeth A Zimmermann, Christoph Riedel, Felix N Schmidt, Kilian E Stockhausen, Yuriy Chushkin, Eric Schaible, Bernd Gludovatz, Eik Vettorazzi, Federico Zontone, Klaus Püschel, Michael Amling, Robert O Ritchie, Björn Busse

Journal Article

ABSTRACTBone fracture risk is influenced by bone quality, which encompasses bones composition as well as its multiscale organization and architecture. Aging and disease deteriorate bone quality, leading to reduced mechanical properties and higher fracture incidence. Largely unexplored is how bone quality and mechanical competence progress during longitudinal bone growth. Human femoral cortical bone was acquired from fetal (n = 1), infantile (n = 3), and 2‐ to 14‐year‐old cases (n = 4) at the mid‐diaphysis. Bone quality was assessed in terms of bone structure, osteocyte characteristics, mineralization, and collagen orientation. The mechanical properties were investigated by measuring tensile deformation at multiple length scales via synchrotron X‐ray diffraction. We find dramatic differences in mechanical resistance with age. Specifically, cortical bone in 2‐ to 14‐year‐old cases exhibits a 160% greater stiffness and 83% higher strength than fetal/infantile cases. The higher mechanical resistance of the 2‐ to 14‐year‐old cases is associated with advantageous bone quality, specifically higher bone volume fraction, better micronscale organization (woven versus lamellar), and higher mean mineralization compared with fetal/infantile cases. Our study reveals that bone quality is superior after remodeling/modeling processes convert the primary woven bone structure to lamellar bone. In this cohort of female children, the microstructural differences at the femoral diaphysis were apparent between the 1‐ to 2‐year‐old cases. Indeed, the lamellar bone in 2‐ to 14‐year‐old cases had a superior structural organization (collagen and osteocyte characteristics) and composition for resisting deformation and fracture than fetal/infantile bone. Mechanistically, the changes in bone quality during longitudinal bone growth lead to higher fracture resistance because collagen fibrils are better aligned to resist tensile forces, while elevated mean mineralization reinforces the collagen scaffold. Thus, our results reveal inherent weaknesses of the fetal/infantile skeleton signifying its inferior bone quality. These results have implications for pediatric fracture risk, as bone produced at ossification centers during childrens longitudinal bone growth could display similarly weak points. © 2019 American Society for Bone and Mineral Research.

Independent Roles of Estrogen Deficiency and Cellular Senescence in the Pathogenesis of Osteoporosis: Evidence in Young Adult Mice and Older Humans

15-08-2019 – Joshua N Farr, Jennifer L Rowsey, Brittany A Eckhardt, Brianne S Thicke, Daniel G Fraser, Tamar Tchkonia, James L Kirkland, David G Monroe, Sundeep Khosla

Journal Article

ABSTRACTEstrogen deficiency is a seminal mechanism in the pathogenesis of osteoporosis. Mounting evidence, however, establishes that cellular senescence, a fundamental mechanism that drives multiple age‐related diseases, also causes osteoporosis. Recently, we systematically identified an accumulation of senescent cells, characterized by increased p16 Ink4a and p21 Cip1 levels and development of a senescence‐associated secretory phenotype (SASP), in mouse bone/marrow and human bone with aging. We then demonstrated that elimination of senescent cells prevented age‐related bone loss using multiple approaches, eg, treating old mice expressing a “suicide” transgene, INK‐ATTAC, with AP20187 to induce apoptosis of p16 Ink4a‐senescent cells or periodically treating old wild‐type mice with “senolytics,” ie, drugs that eliminate senescent cells. Here, we investigate a possible role for estrogen in the regulation of cellular senescence using multiple approaches. First, sex steroid deficiency 2 months after ovariectomy (OVX, n = 15) or orchidectomy (ORCH, n = 15) versus sham surgery (SHAM, n = 15/sex) in young adult (4‐month‐old) wild‐type mice did not alter senescence biomarkers or induce a SASP in bone. Next, in elderly postmenopausal women, 3 weeks of estrogen therapy (n = 10; 74 ± 5 years) compared with no treatment (n = 10; 78 ± 5 years) did not alter senescence biomarkers or the SASP in human bone biopsies. Finally, young adult (4‐month‐old) female INK‐ATTAC mice were randomized (n = 17/group) to SHAM+Vehicle, OVX+Vehicle, or OVX+AP20187 for 2 months. As anticipated, OVX+Vehicle caused significant trabecular/cortical bone loss compared with SHAM+Vehicle. However, treatment with AP20187, which eliminates senescent cells in INK‐ATTAC mice, did not rescue the OVX‐induced bone loss or alter senescence biomarkers. Collectively, our data establish independent roles of estrogen deficiency and cellular senescence in the pathogenesis of osteoporosis, which has important implications for testing novel senolytics for skeletal efficacy, as these drugs will need to be evaluated in preclinical models of aging as opposed to the current FDA model of prevention of OVX‐induced bone loss. © 2019 American Society for Bone and Mineral Research.

Poor Vitamin K Status in Chronic Kidney Disease: An Indirect Indicator of Hip Fragility

15-08-2019 – Toshihiro Sugiyama

Letter

Reply to: Poor Vitamin K Status in Chronic Kidney Disease: An Indirect Indicator of Hip Fragility

15-08-2019 – P Evenepoel, MR Laurent

Letter

Growth Plate Borderline Chondrocytes: A New Source of Metaphyseal Mesenchymal Precursors

15-08-2019 – Eleanor J Mackie

Editorial

Ed Bd, Masthead, Comm List and TOC

15-08-2019 –

Issue Information‐Declaration of Helsinki

15-08-2019 –

Wnt Signaling Inhibits Osteoclast Differentiation by Activating Canonical and Noncanonical cAMP/PKA Pathways

15-08-2019 – Megan M Weivoda, Ming Ruan, Christine M Hachfeld, Larry Pederson, Alan Howe, Rachel A Davey, Jeffrey D Zajac, Yasuhiro Kobayashi, Bart O Williams, Jennifer J Westendorf, Sundeep Khosla, Merry Jo Oursler

Published Erratum

Journal of Bone and Mineral Research: Volume 34, Number 8, August 2019

15-08-2019 –

Nerves in Bone: Evolving Concepts in Pain and Anabolism

15-08-2019 – Jennifer M Brazill, Alec T Beeve, Clarissa S Craft, Jason J Ivanusic, Erica L Scheller

Journal Article, Review

ABSTRACTThe innervation of bone has been described for centuries, and our understanding of its function has rapidly evolved over the past several decades to encompass roles of subtype‐specific neurons in skeletal homeostasis. Current research has been largely focused on the distribution and function of specific neuronal populations within bone, as well as their cellular and molecular relationships with target cells in the bone microenvironment. This review provides a historical perspective of the field of skeletal neurobiology that highlights the diverse yet interconnected nature of nerves and skeletal health, particularly in the context of bone anabolism and pain. We explore what is known regarding the neuronal subtypes found in the skeleton, their distribution within bone compartments, and their central projection pathways. This neuroskeletal map then serves as a foundation for a comprehensive discussion of the neural control of skeletal development, homeostasis, repair, and bone pain. Active synthesis of this research recently led to the first biotherapeutic success story in the field. Specifically, the ongoing clinical trials of anti‐nerve growth factor therapeutics have been optimized to titrated doses that effectively alleviate pain while maintaining bone and joint health. Continued collaborations between neuroscientists and bone biologists are needed to build on this progress, leading to a more complete understanding of neural regulation of the skeleton and development of novel therapeutics. © 2019 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc.

Growth Plate Borderline Chondrocytes Behave as Transient Mesenchymal Precursor Cells

15-08-2019 – Koji Mizuhashi, Mizuki Nagata, Yuki Matsushita, Wanida Ono, Noriaki Ono

Journal Article

ABSTRACTThe growth plate provides a substantial source of mesenchymal cells in the endosteal marrow space during endochondral ossification. The current model postulates that a group of chondrocytes in the hypertrophic zone can escape from apoptosis and transform into cells that eventually become osteoblasts in an area beneath the growth plate. The growth plate is composed of cells with various morphologies; particularly at the periphery of the growth plate immediately adjacent to the perichondrium are “borderline” chondrocytes, which align perpendicularly to other chondrocytes. However, in vivo cell fates of these special chondrocytes have not been revealed. Here we show that borderline chondrocytes in growth plates behave as transient mesenchymal precursor cells for osteoblasts and marrow stromal cells. A single‐cell RNA‐seq analysis revealed subpopulations of Col2a1‐cre
ER‐marked neonatal chondrocytes and their cell type–specific markers. A tamoxifen pulse to Pthrp‐cre
ER mice in the neonatal stage (before the resting zone was formed) preferentially marked borderline chondrocytes. Following the chase, these cells marched into the nascent marrow space, expanded in the metaphyseal marrow, and became Col(2.3 kb)‐GFP+ osteoblasts and Cxcl12‐GFPhigh reticular stromal “CAR” cells. Interestingly, these borderline chondrocyte‐derived marrow cells were short‐lived, as they were significantly reduced during adulthood. These findings demonstrate based on in vivo lineage‐tracing experiments that borderline chondrocytes in the peripheral growth plate are a particularly important route for producing osteoblasts and marrow stromal cells in growing murine endochondral bones. A special microenvironment neighboring the osteogenic perichondrium might endow these chondrocytes with an enhanced potential to differentiate into marrow mesenchymal cells. © 2019 American Society for Bone and Mineral Research.

Explicit Finite Element Models Accurately Predict Subject‐Specific and Velocity‐Dependent Kinetics of Sideways Fall Impact

14-08-2019 – Ingmar Fleps, Pierre Guy, Stephen J Ferguson, Peter A Cripton, Benedikt Helgason

Journal Article

ABSTRACTThe majority of hip fractures in the elderly are the result of a fall from standing or from a lower height. Current injury models focus mostly on femur strength while neglecting subject‐specific loading. This article presents an injury modeling strategy for hip fractures related to sideways falls that takes subject‐specific impact loading into account. Finite element models (FEMs) of the human body were used to predict the experienced load and the femoral strength in a single model. We validated these models for their predicted peak force, effective pelvic stiffness, and fracture status against matching ex vivo sideways fall impacts (n = 11) with a trochanter velocity of 3.1 m/s. Furthermore, they were compared to sideways impacts of volunteers with lower impact velocities that were previously conducted by other groups. Good agreement was found between the ex vivo experiments and the FEMs with respect to peak force (root mean square error RMSE = 10.7%, R2 = 0.85) and effective pelvic stiffness (R2 = 0.92, RMSE = 12.9%). The FEMs were predictive of the fracture status for 10 out of 11 specimens. Compared to the volunteer experiments from low height, the FEMs overestimated the peak force by 25% for low BMI subjects and 8% for high BMI subjects. The effective pelvic stiffness values that were derived from the FEMs were comparable to those derived from impacts with volunteers. The force attenuation from the impact surface to the femur ranged between 27% and 54% and was highly dependent on soft tissue thickness (R2 = 0.86). The energy balance in the FEMS showed that at the time of peak force 79% to 93% of the total energy is either kinetic or was transformed to soft tissue deformation. The presented FEMs allow for direct discrimination between fracture and nonfracture outcome for sideways falls and bridge the gap between impact testing with volunteers and impact conditions representative of real life falls. © 2019 American Society for Bone and Mineral Research.

Fracture and Bone Mineral Density Response by Baseline Risk in Patients Treated With Abaloparatide Followed by Alendronate: Results From the Phase 3 ACTIVExtend Trial

14-08-2019 – Benjamin Leder, Carol Zapalowski, Ming‐yi Hu, Gary Hattersley, Nancy E. Lane, Andrea J. Singer, Robin K. Dore

Journal Article

Abstract
In the randomized, placebo‐controlled, double‐blind phase 3 ACTIVE study (NCT01343004), 18 months of abaloparatide 80 μg daily (subcutaneous injection) in postmenopausal women at risk of osteoporotic fracture significantly reduced the risk of vertebral, nonvertebral, clinical, and major osteoporotic fractures and significantly increased bone mineral density (BMD) versus placebo regardless of baseline risk factors. Women from the abaloparatide and placebo groups who completed ACTIVE were eligible for ACTIVExtend (NCT01657162), in which all enrollees received sequential, open‐label monotherapy with alendronate 70 mg once weekly for up to 24 months. This prespecified analysis evaluated whether fracture risk reductions and BMD gains associated with abaloparatide during ACTIVE persisted through the full 43‐month ACTIVE–ACTIVExtend study period in nine prespecified baseline risk subgroups. Baseline risk subgroups included BMD T‐score at the lumbar spine, total hip, and femoral neck (≤ –2.5 versus > –2.5 and ≤ –3.0 versus > –3.0), history of nonvertebral fracture (yes/no), prevalent vertebral fracture (yes/no), and age (< 65 versus 65 to < 75 versus ≥ 75 years). Forest plots display treatment effect. Treatment‐by‐subgroup interactions were tested using the Breslow‐Day test, Cox proportional hazards model, and ANCOVA model. After the combined ACTIVE–ACTIVExtend study period, reductions in relative risk for new vertebral, nonvertebral, clinical, and major osteoporotic fractures were greater among patients in the abaloparatide/alendronate group than among those in the placebo/alendronate group across all nine baseline risk subgroups. BMD gains at the lumbar spine, total hip, and femoral neck were greater in the abaloparatide/alendronate group versus the placebo/alendronate group. No clinically meaningful interaction between treatment assignment and any baseline risk variable was observed. The sequence of abaloparatide for 18 months followed by alendronate for up to 24 months appears to be an effective treatment option for a wide range of postmenopausal women at risk for osteoporotic fractures.
This article is protected by copyright. All rights reserved.

Reduced Bone Loss Is Associated With Reduced Mortality Risk in Subjects Exposed to Nitrogen Bisphosphonates: A Mediation Analysis

12-08-2019 – Dana Bliuc, Thach Tran, Tineke Geel, Jonathan D Adachi, Claudie Berger, Joop den Bergh, John A Eisman, Piet Geusens, David Goltzman, David A Hanley, Robert Josse, Stephanie Kaiser, Christopher S Kovacs, Lisa Langsetmo, Jerilynn C Prior, Tuan V Nguyen, Jacqueline R Center, for the CaMOS Research Group

Journal Article

ABSTRACTBisphosphonates, potent antiresorptive agents, have been found to be associated with mortality reduction. Accelerated bone loss is, in itself, an independent predictor of mortality risk, but the relationship between bisphosphonates, bone loss, and mortality is unknown. This study aimed to determine whether the association between bisphosphonates and mortality is mediated by a reduction in the rate of bone loss. Participants from the population‐based Canadian Multicentre Osteoporosis Study were followed prospectively between1996 and 2011. Comorbidities and lifestyle factors were collected at baseline and bone mineral density (BMD) at baseline and at years 3 (for those aged 40 to 60 years), 5, and 10. Rate of bone loss was calculated using linear regression. Information on medication use was obtained yearly. Bisphosphonate users grouped into nitrogen bisphosphonates (n
BP; alendronate or risedronate) and etidronate and non‐users (No
Rx) were matched by propensity score, including all baseline factors as well as time of treatment. Coxs proportional hazards models, unadjusted and adjusted for annual rate of bone loss, were used to determine the association between n
BP and etidronate versus No
Rx. For the treatment groups with significant mortality risk reduction, the percent of mortality reduction mediated by a reduction in the rate of bone loss was estimated using a causal mediation analysis. There were 271 pairs of n
BP and matched No
Rx and 327 pairs of etidronate and matched No
Rx. n
BP but not etidronate use was associated with significant mortality risk reduction (hazard ratios HR = 0.61 95% confidence interval 0.39–0.96 and 1.35 95% CI 0.86–2.11 for n
BP and etidronate, respectively). Rapid bone loss was associated with more than 2‐fold increased mortality risk compared with no loss. Mediation analysis indicated that 39% (95% CI 7%–84%) of the n
BP association with mortality was related to a reduction in the rate of bone loss. This finding provides an insight into the mechanism of the relationship between n
BP and survival benefit in osteoporotic patients. © 2019 American Society for Bone and Mineral Research.

Hormone‐independent sexual dimorphism in the regulation of bone resorption by Krox20

09-08-2019 – Elias Sabag, Elinor Halperin, Tamar Liron, Sahar Hiram‐Bab, Baruch Frenkel, Yankel Gabet

Journal Article

ABSTRACTKrox20/EGR2 is a zinc finger transcription factor, implicated in the development of the hindbrain, nerve myelination, and tumor suppression. In skeletal biology, we have demonstrated that Krox20 also regulates adult bone metabolism. We and others have characterized several functions of Krox20 in the osteoclast lineage, namely, preosteoclast proliferation and differentiation, and mature osteoclast apoptosis.
We have previously reported that systemically Krox20‐haploinsufficient mice have a low bone mass with increased bone resorption. However, new data have now revealed that this phenotype is restricted to females. In addition, we discovered that conditional knockout of Krox20 (c
KO) restricted to osteoclasts progenitors is sufficient to induce the same female‐specific bone loss observed in systemic mutants. To test whether this sexual dimorphism results from an interaction between Krox20 and sex hormones, we examined the sex‐ and hormone‐dependent role of Krox20 deficiency on proliferation and apoptosis in osteoclastic cells. Our results indicate that male and female sex hormones (dihydrotestosterone ‐ DHT and estradiol ‐ E2, respectively) as well as Krox20, inhibit preosteoclast proliferation and augment osteoclast apoptosis. The observation that Krox20 expression is inhibited by DHT and E2, negates the hypothesis that the effect of sex hormones is mediated by an increase in Krox20 expression. Interestingly, the effect of Krox20 deficiency was seen only with cells derived from female animals, regardless of any sex hormones added in vitro. In addition, we have identified sexual dimorphism in the expression of several Krox20‐related genes, including NAB2. This sex‐specific epigenetic profile was established at puberty, maintained in the absence of sex hormones, and explains the female‐specific skeletal importance of Krox20. The findings described in this study emphasize the medical importance of sex differences, which may be determined at the epigenetic level.
This article is protected by copyright. All rights reserved.

Artificial Intelligence in Musculoskeletal Imaging: A Paradigm Shift

09-08-2019 – Joseph E. Burns, Jianhua Yao, Ronald M. Summers

Journal Article, Review

Abstract
Artificial intelligence is upending many of our assumptions about the ability of computers to detect and diagnose diseases on medical images. Deep learning, a recent innovation in artificial intelligence, has shown the ability to interpret medical images with sensitivities and specificities at or near that of skilled clinicians for some applications. In this review, we summarize the history of artificial intelligence, present some recent research advances, and speculate about the potential revolutionary clinical impact of the latest computer techniques for bone and muscle imaging.
This article is protected by copyright. All rights reserved.

Association of Bone Density Monitoring in Routine Clinical Practice With Anti‐Osteoporosis Medication Use and Incident Fractures: A Matched Cohort Study

07-08-2019 – William D Leslie, Suzanne N Morin, Patrick Martineau, Mark Bryanton, Lisa M Lix

Journal Article

ABSTRACTRoutine bone mineral density (BMD) monitoring of individuals during the initial 5 years of anti‐osteoporosis treatment is controversial. Using a registry‐based cohort from the Province of Manitoba, Canada, we compared anti‐osteoporosis medication use and fracture outcomes in women with versus without BMD monitoring receiving anti‐osteoporosis medication. We identified 4559 women aged 40 years and older receiving anti‐osteoporosis therapy with serial BMD testing (monitoring) within 5 years (mean interval 3.2 years) and 4559 propensity score–matched women without BMD monitoring. We assessed anti‐osteoporosis medication use over 5 years from a population‐based retail pharmacy database. Incident fractures to 10 years from health services data. During median 10 years observation, 1225 (13.4%) women developed major osteoporotic fracture, including 382 (4.2%) with hip fractures. Monitored women had significantly better fracture‐free survival for major osteoporotic fracture (p = 0.040; 10‐year cumulative risk 1.9% lower, 95% confidence interval CI 0.3–3.6%) and hip fracture (p = 0.001; 10‐year cumulative risk 1.8% lower, 95% CI 0.7–2.8%) compared with women who were not monitored. Hazard ratios (HRs) were significantly lower in monitored versus not monitored women for major osteoporotic fracture (HR = 0.89, 95% CI 0.80–0.98) and hip fracture (HR = 0.74, 95% CI 0.63–0.87). Days of medication use, medication persistence ratio, and treatment switching over 5 years were greater in monitored versus not monitored women. At the end of 5 years, more women in the monitored group persisted on treatment and more switched treatment, with switching behavior associated with an observed interval reduction in BMD. In conclusion, our findings suggest a possible role for BMD monitoring after initiating anti‐osteoporosis therapy in the routine clinical practice setting. © 2019 American Society for Bone and Mineral Research.

Changes in Bone Marrow Adipose Tissue One Year After Roux‐en‐Y Gastric Bypass: A Prospective Cohort Study

07-08-2019 – Ingvild Kristine Blom‐Høgestøl, Tom Mala, Jon A Kristinsson, Ellen‐Margrethe Hauge, Cathrine Brunborg, Hanne Løvdal Gulseth, Erik Fink Eriksen

Journal Article

ABSTRACTBone marrow adipose tissue (BMAT) has been postulated to mediate skeletal fragility in type 2 diabetes (T2D) and obesity. Roux‐en‐Y gastric bypass (RYGB) induces a substantial weight loss and resolution of comorbidities. However, the procedure induces increased bone turnover and fracture rates. No previous study has evaluated biopsy‐measured BMAT fraction preoperatively and after RYGB. In this study, we aimed to investigate BMAT fraction of the hip in participants with and without T2D preoperatively and 1 year after RYGB and explore factors associated with BMAT change. Patients with morbid obesity scheduled for RYGB were examined preoperatively and 1 year after RYGB. Forty‐four participants were included and preoperative examinations were possible in 35. Of these, 33 (94%) met for follow‐up, 2 were excluded, and BMAT estimation was not possible in 1. Eighteen (60%) of the participants were females and 11 (37%) had T2D. Preoperative BMAT fraction was positively associated with glycosylated hemoglobin and negatively associated with areal bone mineral density (a
BMD). After RYGB, BMAT fraction decreased from 40.4 ± 1.7% to 35.6 ± 12.8%, p = 0.042, or with mean percent change of 10.7% of preoperative BMAT fraction. Change in BMAT fraction was positively associated with change in body mass index (BMI) and total body fat. In females, we observed a mean percent reduction of 22.4 ± 19.6%, whereas in males BMAT increased with a mean percent of 6.8 ± 37.5%, p = 0.009. For males, changes in estradiol were associated with BMAT change; this was not observed for females. In participants with and without T2D, the mean percent BMAT reduction was 5.8 ± 36.9% and 13.5 ± 28.0%, respectively, p = 0.52. We conclude that a high BMAT seems to be associated with lower a
BMD and poorer glycemic control in obese subjects. After RYGB, we observed a significant decrease in BMAT. The reduction in BMAT did not differ between participants with and without T2D, but appeared sex specific. © 2019 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.

Denosumab Improves Glomerular Filtration Rate in Osteoporotic Patients With Normal Kidney Function by Lowering Serum Phosphorus

07-08-2019 – Daichi Miyaoka, Masaaki Inaba, Yasuo Imanishi, Noriyuki Hayashi, Masaya Ohara, Yuki Nagata, Masafumi Kurajoh, Shinsuke Yamada, Katsuhito Mori, Masanori Emoto

Journal Article

ABSTRACTHigher serum phosphorus (Pi) increases the risk for chronic kidney disease (CKD). It was reported that a single administration of denosumab or zoledronate significantly suppressed serum Pi levels as well as those of bone resorption markers in serum. Also, previous evidences suggest a link between bone anti‐resorptive therapy and vasoprotective/renoprotective effects through mechanisms that remain unexplored. The aim of this study is to assess the renoprotective effect of denosumab and involvement of denosumab‐induced reduction in serum Pi in osteoporotic patients. Osteoporotic patients (n = 73) without overt proteinuria in dipstick test results were treated with denosumab (60 mg) every 6 months during the study period (24 months). Estimated glomerular filtration rate based on serum cystatin C (e
GFRcys) was used as a filtration marker and tartrate‐resistant acid phosphatase‐5b (TRACP‐5b) as a bone resorption marker. For analysis of non‐CKD patients (n = 56), those with e
GFRcys <60 m
L/min/1.73 m2 were excluded. A single injection of denosumab suppressed serum Pi as well as TRACP‐5b during the first 6 months, whereas age‐related decline in e
GFRcys was significantly reversed, with an increase of 2.75 ± 1.2 m
L/min/1.73 m2 after 24 months noted. Multivariate analysis showed that serum Pi reduction following the initial denosumab injection was positively associated with serum TRACP‐5b suppression during that same period (β = 0.241, p = 0.049). In addition, a positive association of serum Pi suppression, but not of corrected calcium or TRACP‐5b, with e
GFRcys increase after 24 months (β = 0.321, p = 0.014) was found after adjustments for gender, age, BMI, antihypertensive drug use, albumin, and e
GFRcys. The same was observed in osteoporotic cases restricted to non‐CKD patients. In conclusion, serum Pi reduction resulting from phosphorus load decrement from bone induced by denosumab is a determinant for e
GFRcys increase. Early introduction of bone antiresorptive therapy can retain glomerular filtration in osteoporosis cases, including non‐CKD patients. © 2019 American Society for Bone and Mineral Research.

The Design and Validation of a New Algorithm to Identify Incident Fractures in Administrative Claims Data

05-08-2019 – Nicole C Wright, Shanette G Daigle, Mary E Melton, Elizabeth S Delzell, Akhila Balasubramanian, Jeffrey R Curtis

Journal Article, Review

ABSTRACTOur study validated a claims‐based algorithm for the identification of incident and recurrent fractures in administrative data. We used Centers for Medicare and Medicaid (CMS) claims from 2005 to 2014 linked to the Reasons for Geographic and Racial Differences in Stroke (REGARDS) database. Case qualifying (CQ) fractures were identified among participants with ≥12 months of fee‐for‐service coverage before first fracture claim and ≥6 months after. Recurrent fractures were defined as the first CQ fracture that occurred following a clean period of at least 90 days from the last claim associated with the preceding incident fracture. We used medical records (discharge summary, imaging, and surgical report) to adjudicate fractures. We calculated positive predictive values (PPVs) for incident and recurrent fractures. Our study was not designed to assess the algorithm sensitivity or negative predictive value. We identified 2049 potential incident fractures from claims among 1650 participants. Record retrieval was attempted for 728 (35.5%) suspected incident fractures (prioritizing more recent CQ fractures associated with osteoporosis, but without explicitly requiring any osteoporosis ICD‐9 diagnosis code). Our final sample included 520 claims‐identified fractures with medical records, of which 502 (96.5%) were confirmed. The PPVs (95% CI) of the hip, wrist, humerus, and clinical vertebra—all exceeded 95%. We identified 117 beneficiaries with 292 ≥2 CQ fracture episodes at the same site, and attempted retrieval on 105 (36.0%) episodes. Our analytic sample included 72 (68.5%) CQ episodes from 33 participants. The PPVs for identifying recurrent clinical vertebral, hip/femur, and nonvertebral fractures with a 90‐day clean period exceeded 95%. Although we could not ascertain sensitivity, our updated fracture identification algorithms had high PPV for the identification of incident and recurrent fractures of the same site. Although medical record review and clinical adjudication remain a gold standard, our claims‐based algorithm provides an alternative approach to fracture ascertainment when high PPV is desired. © 2019 American Society for Bone and Mineral Research.

TNFRSF11A‐Associated Dysosteosclerosis: A Report of the Second Case and Characterization of the Phenotypic Spectrum

05-08-2019 – Jing‐yi Xue, Zheng Wang, Satoshi Shinagawa, Hirofumi Ohashi, Nao Otomo, Nursel H Elcioglu, Tomoki Nakashima, Gen Nishimura, Shiro Ikegawa, Long Guo

Journal Article

ABSTRACTDysosteosclerosis (DOS) is a distinct form of sclerosing bone disease characterized by irregular osteosclerosis and platyspondyly. DOS is genetically heterogeneous; however, only five cases with SLC29A3 mutations and a single case with a splice‐site mutation of TNFRSF11A have been reported, and TNFRSF11A is also a causal gene for osteopetrosis, autosomal recessive 7 (OP‐AR7). Thus, the causal genes of DOS and their genotype‐phenotype associations remain unclear. In this study, we examined a Japanese patient with DOS and found a novel variant in TNFRSF11A. The homozygous variant was a G to T transversion at the first nucleotide of exon 9 (c.784G>T). Although the variant was predicted to cause a stop codon mutation (p.
E262*), in silico evaluation of the exonic splicing elements followed by RT‐PCR for the patient‐derived cells showed that it caused aberrant splicing due to the change in the exonic splicing element and produced two types of aberrant transcripts: One caused a premature stop codon (p.
E262Vfs*17) leading to nonsense mutation‐mediated m
RNA decay; the other produced a protein with interstitial deletion (p.
E262_Q279del). The effects of the mutation on five splicing isoforms of TNFRSF11A were different from those in OP‐AR7, but comparable with those in the first DOS with the TNFRSF11A mutation. Thus, we identified the second case of DOS caused by the TNFRSF11A splice‐site mutation and confirmed the novel disease entity. © 2019 American Society for Bone and Mineral Research.

Expression of a Degradation‐Resistant β‐Catenin Mutant in Osteocytes Protects the Skeleton From Mechanodeprivation‐Induced Bone Wasting

05-08-2019 – Whitney A Bullock, April M Hoggatt, Daniel J Horan, Karl J Lewis, Hiroki Yokota, Steven Hann, Matthew L Warman, Aimy Sebastian, Gabriela G Loots, Fredrick M Pavalko, Alexander G Robling

Journal Article

ABSTRACTMechanical stimulation is a key regulator of bone mass, maintenance, and turnover. Wnt signaling is a key regulator of mechanotransduction in bone, but the role of β‐catenin—an intracellular signaling node in the canonical Wnt pathway—in disuse mechanotransduction is not defined. Using the β‐catenin exon 3 flox (constitutively active CA) mouse model, in conjunction with a tamoxifen‐inducible, osteocyte‐selective Cre driver, we evaluated the effects of degradation‐resistant β‐catenin on bone properties during disuse. We hypothesized that if β‐catenin plays an important role in Wnt‐mediated osteoprotection, then artificial stabilization of β‐catenin in osteocytes would protect the limbs from disuse‐induced bone wasting. Two disuse models were tested: tail suspension, which models fluid shift, and botulinum‐toxin (botox)‐induced muscle paralysis, which models loss of muscle force. Tail suspension was associated with a significant loss of tibial bone mass and density, reduced architectural properties, and decreased bone formation indices in uninduced (control) mice, as assessed by dual‐energy X‐ray absorptiometry (DXA), micro‐computed tomography (µCT), and histomorphometry. Activation of the βcat
CA allele in tail‐suspended mice resulted in little to no change in those properties; ie, these mice were protected from bone loss. Similar protective effects were observed among botox‐treated mice when the βcat
CA was activated. RNAseq analysis of altered gene regulation in tail‐suspended mice yielded 35 genes, including Wnt11, Gli1, Nell1, Gdf5, and Pgf, which were significantly differentially regulated between tail‐suspended β‐catenin stabilized mice and tail‐suspended nonstabilized mice. Our findings indicate that selectively targeting/blocking of β‐catenin degradation in bone cells could have therapeutic implications in mechanically induced bone disease. © 2019 American Society for Bone and Mineral Research.

Parathyroid Hormone Shifts Cell Fate of a Leptin Receptor‐Marked Stromal Population from Adipogenic to Osteoblastic Lineage

02-08-2019 – Mengyu Yang, Atsushi Arai, Nobuyuki Udagawa, Lijuan Zhao, Daisuke Nishida, Kohei Murakami, Toru Hiraga, Ryoko Takao‐Kawabata, Koichi Matsuo, Toshihisa Komori, Yasuhiro Kobayashi, Naoyuki Takahashi, Yukihiro Isogai, Toshinori Ishizuya, Akira Yamaguchi, Toshihide Mizoguchi

Journal Article

ABSTRACTIntermittent parathyroid hormone (i
PTH) treatment induces bone anabolic effects that result in the recovery of osteoporotic bone loss. Human PTH is usually given to osteoporotic patients because it induces osteoblastogenesis. However, the mechanism by which PTH stimulates the expansion of stromal cell populations and their maturation toward the osteoblastic cell lineage has not be elucidated. Mouse genetic lineage tracing revealed that i
PTH treatment induced osteoblastic differentiation of bone marrow (BM) mesenchymal stem and progenitor cells (MSPCs), which carried the leptin receptor (Lep
R)‐Cre. Although these findings suggested that part of the PTH‐induced bone anabolic action is exerted because of osteoblastic commitment of MSPCs, little is known about the in vivo mechanistic details of these processes. Here, we showed that Lep
R+MSPCs differentiated into type I collagen (Col1)+ mature osteoblasts in response to i
PTH treatment. Along with osteoblastogenesis, the number of Col1+ mature osteoblasts increased around the bone surface, although most of them were characterized as quiescent cells. However, the number of Lep
R‐Cre‐marked lineage cells in a proliferative state also increased in the vicinity of bone tissue after i
PTH treatment. The expression levels of SP7/osterix (Osx) and Col1, which are markers for osteoblasts, were also increased in the Lep
R+MSPCs population in response to i
PTH treatment. In contrast, the expression levels of Cebpb, Pparg, and Zfp467, which are adipocyte markers, decreased in this population. Consistent with these results, i
PTH treatment inhibited 5‐fluorouracil‐ or ovariectomy (OVX)‐induced Lep
R+MSPC‐derived adipogenesis in BM and increased Lep
R+MSPC‐derived osteoblasts, even under the adipocyte‐induced conditions. Treatment of OVX rats with i
PTH significantly affected the osteoporotic bone tissue and expansion of the BM adipose tissue. These results indicated that i
PTH treatment induced transient proliferation of the Lep
R+MSPCs and skewed their lineage differentiation from adipocytes toward osteoblasts, resulting in an expanded, quiescent, and mature osteoblast population. © 2019 American Society for Bone and Mineral Research.

Specific RANK Cytoplasmic Motifs Drive Osteoclastogenesis

02-08-2019 – Yuyu Li, Zhenqi Shi, Joel Jules, Shenyuan Chen, Robert A Kesterson, Dongfeng Zhao, Ping Zhang, Xu Feng

Journal Article

ABSTRACTUpon receptor activator of NF‐κB ligand (RANKL) binding, RANK promotes osteoclast formation through the recruitment of tumor necrosis factor (TNF) receptor‐associated factors (TRAFs). In vitro assays identified two RANK intracellular motifs that bind TRAFs: PVQEET560–565 (Motif 2) and PVQEQG604–609 (Motif 3), which potently mediate osteoclast formation in vitro. To validate the in vitro findings, we have generated knock‐in (KI) mice harboring inactivating mutations in RANK Motifs 2 and 3. Homozygous KI (RANKKI/KI) mice are born at the predicted Mendelian frequency and normal in tooth eruption. However, RANKKI/KI mice exhibit significantly more trabecular bone mass than age‐ and sex‐matched heterozygous KI (RANK+/KI) and wild‐type (RANK+/+) counterparts. Bone marrow macrophages (BMMs) from RANKKI/KI mice do not form osteoclasts when they are stimulated with macrophage colony‐stimulating factor (M‐CSF) and RANKL in vitro. RANKL is able to activate the NF‐κB, ERK, p38, and JNK pathways in RANKKI/KI BMMs, but it cannot stimulate c‐Fos or NFATc1 in the RANKKI/KI cells. Previously, we showed that RANK signaling plays an important role in Porphyromonas gingivalis (Pg)‐mediated osteoclast formation by committing BMMs into the osteoclast lineage. Here, we show that RANKL‐primed RANKKI/KI BMMs are unable to differentiate into osteoclasts in response to Pg stimulation, indicating that the two RANK motifs are required for Pg‐induced osteoclastogenesis. Mechanistically, RANK Motifs 2 and 3 facilitate Pg‐induced osteoclastogenesis by stimulating c‐Fos and NFATc1 expression during the RANKL pretreatment phase as well as rendering c‐Fos and NFATc1 genes responsive to subsequent Pg stimulation. Cell‐penetrating peptides (CPPs) conjugated with RANK segments containing Motif 2 or 3 block RANKL‐ and Pg‐mediated osteoclastogenesis. The CPP conjugates abrogate RANKL‐stimulated c‐Fos and NFATc1 expression but do not affect RANKL‐induced activation of NF‐κB, ERK, p38, JNK, or Akt signaling pathway. Taken together, our current findings demonstrate that RANK Motifs 2 and 3 play pivotal roles in osteoclast formation in vivo and mediate Pg‐induced osteoclastogenesis in vitro.

Cytokine‐ and stretch‐induced sphingosine1‐phosphate production by enthesis cells could favor abnormal ossification in spondyloarthritis

02-08-2019 – Alaeddine El Jamal, Anne Briolay, Saida Mebarek, Benoit Le Goff, Frédéric Blanchard, David Magne, Leyre Brizuela, Carole Bougault

Journal Article

ABSTRACTSpondyloarthritis (Sp
A) is a common rheumatic disease characterized by enthesis inflammation (enthesitis) and ectopic ossification (enthesophytes). The current pathogenesis model suggests that inflammation and mechanical stress are both strongly involved in Sp
A pathophysiology. We have previously observed that the levels of sphingosine 1‐phosphate (S1P), a bone anabolic molecule, were particularly high in Sp
A patients serum compared to healthy donors. Therefore, we wondered how this deregulation was related to Sp
A molecular mechanisms. Mouse primary osteoblasts, chondrocytes, and tenocytes were used as cell culture models. The sphingosine kinase 1 (Sphk1) gene expression and S1P secretion were significantly enhanced by cyclic stretch in osteoblasts and chondrocytes. Further, TNF‐α and IL‐17, cytokines implicated in enthesitis, increased Sphk1 m
RNA in chondrocytes in an additive manner when combined to stretch. The immunochemistry on mouse ankles showed that SK1 was localized in some chondrocytes; the addition of a pro‐inflammatory cocktail augmented Sphk1 expression in cultured ankles. Subsequently, Fingolimod was used to block S1P metabolism in cell cultures. It inhibited S1P receptors (S1PRs) signaling and SK1 and SK2 activity in both osteoblasts and chondrocytes. Fingolimod also reduced S1PR‐induced activation by Sp
A patients synovial fluid (SF), demonstrating that the stimulation of chondrocytes by SFs from Sp
A patients involves S1P. In addition, when the osteogenic culture medium was supplemented with Fingolimod, alkaline phosphatase activity, matrix mineralization, and bone formation markers were significantly reduced in osteoblasts and hypertrophic chondrocytes. Osteogenic differentiation was accompanied by an increase in S1prs m
RNA, especially S1P1/3, but their contribution to S1P‐impact on mineralization seemed limited. Our results suggest that S1P might be overproduced in Sp
A enthesis in response to cytokines and mechanical stress, most likely by chondrocytes. Moreover, S1P could locally favor the abnormal ossification of the enthesis; therefore, blocking the S1P metabolic pathway could be a potential therapeutic approach for the treatment of Sp
A.
This article is protected by copyright. All rights reserved.

Lymphatic Endothelial Cells Produce M‐CSF, Causing Massive Bone Loss in Mice

02-08-2019 – Wensheng Wang, Hua Wang, Xichao Zhou, Xing Li, Wen Sun, Michael Dellinger, Brendan F Boyce, Lianping Xing

Mendelian Randomization Analysis Reveals a Causal Influence of Circulating Sclerostin Levels on Bone Mineral Density and Fractures

02-08-2019 – Jie Zheng, Winfried Maerz, Ingrid Gergei, Marcus Kleber, Christiane Drechsler, Christoph Wanner, Vincent Brandenburg, Sjur Reppe, Kaare M Gautvik, Carolina Medina‐Gomez, Enisa Shevroja, Arthur Gilly, Young‐Chan Park, George Dedoussis, Eleftheria Zeggini, Mattias Lorentzon, Petra Henning, Ulf H Lerner, Karin H Nilsson, Sofia Movérare‐Skrtic, Denis Baird, Benjamin Elsworth, Louise Falk, Alix Groom, Terence D Capellini, Elin Grundberg, Maria Nethander, Claes Ohlsson, George Davey Smith, Jonathan H Tobias

Journal Article

ABSTRACTIn bone, sclerostin is mainly osteocyte‐derived and plays an important local role in adaptive responses to mechanical loading. Whether circulating levels of sclerostin also play a functional role is currently unclear, which we aimed to examine by two‐sample Mendelian randomization (MR). A genetic instrument for circulating sclerostin, derived from a genomewide association study (GWAS) meta‐analysis of serum sclerostin in 10,584 European‐descent individuals, was examined in relation to femoral neck bone mineral density (BMD; n = 32,744) in GEFOS and estimated bone mineral density (e
BMD) by heel ultrasound (n = 426,824) and fracture risk (n = 426,795) in UK Biobank. Our GWAS identified two novel serum sclerostin loci, B4GALNT3 (standard deviation SD) change in sclerostin per A allele (β = 0.20, p = 4.6 × 10−49) and GALNT1 (β  = 0.11 per G allele, p = 4.4 × 10−11). B4GALNT3 is an N‐acetyl‐galactosaminyltransferase, adding a terminal Lacdi
NAc disaccharide to target glycocoproteins, found to be predominantly expressed in kidney, whereas GALNT1 is an enzyme causing mucin‐type O‐linked glycosylation. Using these two single‐nucleotide polymorphisms (SNPs) as genetic instruments, MR revealed an inverse causal relationship between serum sclerostin and femoral neck BMD (β = –0.12, 95% confidence interval CI –0.20 to –0.05) and e
BMD (β = –0.12, 95% CI –0.14 to –0.10), and a positive relationship with fracture risk (β = 0.11, 95% CI 0.01 to 0.21). Colocalization analysis demonstrated common genetic signals within the B4GALNT3 locus for higher sclerostin, lower e
BMD, and greater B4GALNT3 expression in arterial tissue (probability >99%). Our findings suggest that higher sclerostin levels are causally related to lower BMD and greater fracture risk. Hence, strategies for reducing circulating sclerostin, for example by targeting glycosylation enzymes as suggested by our GWAS results, may prove valuable in treating osteoporosis. © 2019 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc.

Burosumab Improved Histomorphometric Measures of Osteomalacia in Adults with X‐Linked Hypophosphatemia: A Phase 3, Single‐Arm, International Trial

01-08-2019 – Karl L. Insogna, Frank Rauch, Peter Kamenický, Nobuaki Ito, Takuo Kubota, Akie Nakamura, Lin Zhang, Matt Mealiffe, Javier San Martin, Anthony A. Portale

Journal Article

ABSTRACTIn adults with X‐linked hypophosphatemia (XLH), excess FGF23 impairs renal phosphate reabsorption and suppresses production of 1,25‐dihydroxyvitamin D, resulting in chronic hypophosphatemia and persistent osteomalacia. Osteomalacia is associated with poor bone quality causing atraumatic fractures, pseudofractures, delayed fracture healing, and bone pain. Burosumab is a fully human monoclonal antibody against FGF23. UX023‐CL304 is an ongoing, open‐label, single‐arm, Phase 3 study investigating the efficacy of subcutaneous burosumab, 1.0 mg/kg administered every 4 weeks, in improving osteomalacia in adults with XLH who have not been treated for at least two years before enrollment. The primary endpoint was improvement in osteoid volume/bone volume assessed by transiliac bone biopsies obtained at Baseline and Week 48. Additional assessments included serum phosphorus, markers of bone turnover, fracture/pseudofracture healing, and safety. Fourteen subjects enrolled, 13 completed 48 weeks, and 11 completed paired biopsies. All osteomalacia‐related histomorphometric measures improved significantly at Week 48 (mean percent change: osteoid volume/bone volume − 54%, osteoid thickness − 32%, osteoid surface/bone surface −26%, median mineralization lag time − 83%). Mean serum phosphorus concentration averaged across the mid‐point of the dose cycle between Weeks 0–24 was 3.3 mg/d
L, a 50% increase from 2.2 mg/d
L at Baseline. Markers of bone formation and resorption increased at Week 48 (LS mean increase: P1NP +77%, CTx +36%; both p < 0.0001). All subjects had ≥1 treatment‐emergent adverse event (AE). Most AEs were mild to moderate in severity. Two subjects experienced serious AEs (migraine; paresthesia) that were unrelated to treatment and resolved. Eleven subjects had 18 biopsy procedure‐related AEs: 14 for pain, two for itch, and one each for headache and bandage irritation. No deaths or incidents of hyperphosphatemia occurred. In conclusion, by normalizing phosphate homeostasis, burosumab significantly improved osteomalacia in adults with XLH, which likely explains the improved fracture healing and amelioration of skeletal complications.
This article is protected by copyright. All rights reserved.

A New 1,25 Dihydroxy Vitamin D Analog with Strong Bone Anabolic Activity in OVX Rats with Little or No Bone Resorptive Activity

01-08-2019 – Lori A. Plum, Julia Zella, Margaret Clagett‐Dame, Hector F. DeLuca

Journal Article

ABSTRACTA new 1α,25‐dihydroxy vitamin D₃ analog (2‐methylene‐22(E)‐(24R)‐22‐dehydro‐1α,24,25‐trihydroxy‐19‐norvitamin D3 or WT‐51) has been tested as a possible therapeutic for osteoporosis. It is 1/10th as active as 1,25(OH)2D3 in binding affinity for the vitamin D receptor but is at least 200 times more active than 1,25(OH)2D3 and equal to that of 2MD (2‐methylene‐19‐nor‐(20S)‐1α,25(OH)2D3, an analog previously tested in postmenopausal women), in supporting bone formation by isolated osteoblasts in culture. However, in contrast to 2MD, it is virtually inactive on bone resorption in vivo. WT‐51 markedly increased bone mass (lumbar and femur) in ovariectomized female rats. Further, bone strength tested by the 3‐point bending system is significantly increased by WT‐51. Thus, WT‐51 is an attractive candidate for the treatment of post‐menopausal osteoporosis.
This article is protected by copyright. All rights reserved.

Vertebral Fracture Assessment Increases Use of Pharmacologic Therapy for Fracture Prevention in Clinical Practice

01-08-2019 – John T. Schousboe, Lisa M. Lix, Suzanne N. Morin, Sheldon Derkatch, Mark Bryanton, Mashael Alhrbi, William D. Leslie

Journal Article

ABSTRACTThe impact of vertebral fracture assessment on lateral spine images (VFA) in clinical practice on subsequent patient use of fracture prevention medication is unknown. Our objective was to determine the association of prevalent vertebral fracture identified on bone density lateral spine images (positive VFA) with subsequent use of fracture prevention therapy in usual clinical practice, using the Manitoba Bone Density Program database prospective observational cohort. Since 2010, targeted VFA imaging has been done at the time of bone densitometry in Manitoba for 21% of women and men meeting criteria based on age, bone mineral density, height loss, and glucocorticoid use. Among 6652 treatment naive individuals with at least 90 days follow up who had VFA imaging, 923 (13.9%) had one or more definite vertebral fractures identified using a modified ABQ method. For those with a positive VFA, their bone density reports stated the patient was at high risk of subsequent fracture and qualified for fracture prevention therapy. Subsequent osteoporosis treatment initiated within the next 12 months was identified using population‐based pharmacy data. Logistic regression models were used to estimate the association of positive VFA with subsequent Rx, compared to negative VFA. Fracture prevention medication was started by 2127 (32%) individuals, 52.3% with positive vs 28.4% with negative VFA (p‐value<0.001). This association was substantially stronger in those designated (before VFA results were known) to have low or moderate fracture risk compared to high fracture risk (interaction p‐value <0.001), and in those with osteopenia (OR 4.51, 95% CI 3.48 to 5.85) compared to those with osteoporosis by BMD criteria (OR 1.72, 95% CI 1.43 to 2.08, interaction p‐value <0.001). Targeted VFA imaging at the time of bone densitometry substantially improves identification of those at high fracture risk and fracture prevention medication use amongst those with prevalent vertebral fracture.
This article is protected by copyright. All rights reserved.

Low Vitamin D Status Is Associated With Impaired Bone Quality and Increased Risk of Fracture‐Related Hospitalization in Older Australian Women

01-08-2019 – Kun Zhu, Joshua R Lewis, Marc Sim, Richard L Prince

Journal Article

ABSTRACTThe vitamin D debate relates in part to ideal public health population levels of circulating 25‐hydroxyvitamin D (25OHD) to maintain bone structure and reduce fracture. In a secondary analysis of 1348 women aged 70 to 85 years at baseline (1998) from the Perth Longitudinal Study of Aging in Women (a 5‐year calcium supplementation trial followed by two 5‐year extensions), we examined the dose‐response relations of baseline plasma 25OHD with hip DXA BMD at year 1, lumbar spine BMD, and trabecular bone score (TBS) at year 5, and fracture‐related hospitalizations over 14.5 years obtained by health record linkage. Mean baseline plasma 25OHD was 66.9 ± 28.2 nmol/L and 28.5%, 36.4%, and 35.1% of women had levels <50, 50 to 74.9, and ≥75 nmol/L, respectively. Generalized additive models showed that total hip and femoral neck BMD and TBS, but not spine BMD, were higher with increasing plasma 25OHD up to 100 nmol/L. Compared with those with 25OHD <50 nmol/L, women with 25OHD ≥75 nmol/L had significantly higher total hip and femoral neck BMD at year 1 (3.3% to 3.9%) and TBS at year 5 (2.0%), all P < 0.05. During the follow‐up, 27.6% of women experienced any fracture‐related hospitalization and 10.6% hip fracture‐related hospitalization. Penalized spline regression models showed a decrease in risk with increased 25OHD levels up to 65 nmol/L and 75 nmol/L for hip fracture and any fracture‐related hospitalization, respectively. Cox regression grouped analyses showed that compared with women with 25OHD <50 nmol/L, those with 25OHD levels 50 to 74.9 and ≥75 nmol/L had significantly lower risk for hip fracture HR 0.60 (95% CI, 0.40 to 0.91) and 0.61 (95% CI, 0.40 to 0.92), respectively, and any fracture‐related hospitalization HR 0.77 (95% CI, 0.59 to 0.99) and 0.70 (95% CI, 0.54 to 0.91), respectively. In older white women, 25OHD levels >50 nmol/L are a minimum public health target and 25OHD levels beyond 75 nmol/L may not have additional benefit to reduce fracture risk. © 2019 American Society for Bone and Mineral Research.

VEGFA From Early Osteoblast Lineage Cells (Osterix+) Is Required in Mice for Fracture Healing

01-08-2019 – Evan G Buettmann, Jennifer A McKenzie, Nicole Migotsky, David AW Sykes, Pei Hu, Susumu Yoneda, Matthew J Silva

Journal Article

ABSTRACTBone formation via intramembranous and endochondral ossification is necessary for successful healing after a wide range of bone injuries. The pleiotropic cytokine, vascular endothelial growth factor A (VEGFA) has been shown, via nonspecific pharmacologic inhibition, to be indispensable for angiogenesis and ossification following bone fracture and cortical defect repair. However, the importance of VEGFA expression by different cell types during bone healing is not well understood. We sought to determine the role of VEGFA from different osteoblast cell subsets following clinically relevant models of bone fracture and cortical defect. Ubiquitin C (UBC), Osterix (Osx), or Dentin matrix protein 1 (Dmp1) Cre‐ERT2 mice (male and female) containing floxed VEGFA alleles (VEGFAfl/fl) were either given a femur full fracture, ulna stress fracture, or tibia cortical defect at 12 weeks of age. All mice received tamoxifen continuously starting 2 weeks before bone injury and throughout healing. UBC Cre‐ERT2 VEGFAfl/fl (UBC c
KO) mice, which were used to mimic nonspecific inhibition, had minimal bone formation and impaired angiogenesis across all bone injury models. UBC c
KO mice also exhibited impaired periosteal cell proliferation during full fracture, but not stress fracture repair. Osx Cre‐ERT2 VEGFAfl/fl (Osx c
KO) mice, but not Dmp1 Cre‐ERT2 VEGFAfl/fl (Dmp1 c
KO) mice, showed impaired periosteal bone formation and angiogenesis in models of full fracture and stress fracture. Neither Osx c
KO nor Dmp1 c
KO mice demonstrated significant impairments in intramedullary bone formation and angiogenesis following cortical defect. These data suggest that VEGFA from early osteolineage cells (Osx+), but not mature osteoblasts/osteocytes (Dmp1+), is critical at the time of bone injury for rapid periosteal angiogenesis and woven bone formation during fracture repair. Whereas VEGFA from another cell source, not from the osteoblast cell lineage, is necessary at the time of injury for maximum cortical defect intramedullary angiogenesis and osteogenesis. © 2019 American Society for Bone and Mineral Research.

The Effect of a Screening and Treatment Program for the Prevention of Fractures in Older Women: A Randomized Pragmatic Trial

01-08-2019 – Thomas Merlijn, Karin MA Swart, Natasja M Schoor, Martijn W Heymans, Babette C Zwaard, Amber A Heijden, Femke Rutters, Paul Lips, Henriëtte E Horst, Christy Niemeijer, J Coen Netelenbos, Petra JM Elders

Journal Article

ABSTRACTPopulation screening for fracture risk may reduce the fracture incidence. In this randomized pragmatic trial, the SALT Osteoporosis Study (SOS), we studied whether screening for fracture risk and subsequent treatment in primary care can reduce fractures compared with usual care. A total of 11,032 women aged 65 to 90 years with ≥1 clinical risk factor for fractures were individually randomized to screening (n = 5575) or usual care (n = 5457). Participants in the screening group underwent a screening program, including bone densitometry and vertebral fracture assessment. Participants with a high 10‐year fracture probability (FRAX) or a vertebral fracture were offered treatment with anti‐osteoporosis medication by their general practitioner. Incident fractures as reported by questionnaires were verified with medical records. Follow‐up was completed by 94% of the participants (mean follow‐up = 3.7 years). Of the 5575 participants in the screening group, 1417 (25.4%) had an indication for anti‐osteoporosis medication. Screening and subsequent treatment had no statistically significant effect on the primary outcome fracture (hazard ratio HR = 0.97; 95% confidence interval CI 0.87–1.08), nor on the secondary outcomes osteoporotic fractures (HR = 0.91; 95% CI 0.81–1.03), major osteoporotic fractures (HR = 0.91; 95% CI 0.80–1.04), hip fractures (HR = 0.91; 95% CI 0.71–1.15), falls (odds ratio OR = 0.91; 95% CI 0.72–1.15), or mortality (HR = 1.03; 95% CI 0.91–1.17). Post hoc explorative finding suggested that screening might be most effective after a recent fracture (HR = 0.65; 95% CI 0.44–0.96 for major osteoporotic fractures and HR = 0.38; 95% CI 0.18–0.79 for hip fractures). The results of this study might have been compromised by nonparticipation and medication nonadherence in the screening group. Overall, this study does not provide sufficient indications to consider screening for fracture prevention. However, we cannot exclude its clinical relevance to reduce (major) osteoporotic fractures and hip fractures because of the relatively small number of women with a treatment indication in the intervention group. © 2019 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.

Increased risk of bone fractures in hemodialysis patients treated with proton pump inhibitors in real world: results from the Dialysis Outcomes and Practice Patterns Study (DOPPS)

31-07-2019 – “Maria Fusaro, Graziella DArrigo, Annalisa Pitino, Giorgio Iervasi, Francesca Tentori, Bruce Robinson, Andrea Aghi, Brian Bieber, Keith Mccullogh, Fabrizio Fabris, Mario Plebani, Sandro Giannini, Maurizio Gallieni, Giovanni Tripepi”

Journal Article

ABSTRACTLong‐term treatment with Proton Pump Inhibitors (PPIs) is associated with an increased risk of fractures in the general population. PPIs are widely prescribed to dialysis patients but to date no study specifically tested, by state‐of‐art statistical methods, the relationship between PPIs use and fractures in this patient‐population. This study aimed to assess whether PPIs use is associated with bone fractures (i.e. hip fractures and fractures other than hip fractures) in a large international cohort of hemodialysis patients. We considered an observational prospective cohort of 27097 hemodialysis patients from the DOPPS study. Data analysis was performed by the Fine & Gray method, considering the competitive risk of mortality, as well as by a cause‐specific hazards Cox model dealing death as a censoring event and matching patients according to the prescription time. Out of 27,097 hemodialysis patients, 13,283 patients (49%) were on PPI treatment. Across the follow‐up (median:19 months), 3.8 bone fractures x 100 person‐years and 1.2 hip fractures x 100 person‐years occurred. In multiple Cox models, considering the competitive risk of mortality, the incidence rate of bone (SHR: 1.22, 95% CI: 1.10–1.36, P < 0.001) and hip fractures (SHR: 1.35, 95% CI: 1.13–1.62, P = 0.001) was significantly higher in PPI treated than in PPI untreated patients. These findings held true also in multiple, cause‐specific, hazards Cox models matching patients according to the prescription time (bone fractures, HR: 1.47, 95% CI: 1.23–1.76, P < 0.001, hip fractures (HR: 1.85, 95% CI: 1.37–2.50, P < 0.001). The use of PPIs requires caution and a careful evaluation of risks/benefits ratio in hemodialysis patients.
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Conditional Activation of NF‐κB Inducing Kinase (NIK) in the Osteolineage Enhances Both Basal and Loading‐Induced Bone Formation

31-07-2019 – Jennifer L Davis, Linda Cox, Christine Shao, Cheng Lyu, Shaopeng Liu, Rajeev Aurora, Deborah J Veis

Journal Article

ABSTRACTStudies from global loss‐of‐function mutants suggest that alternative NF‐κB downstream of NF‐κB inducing kinase (NIK) is a cell‐intrinsic negative regulator of osteogenesis. However, the interpretation of the osteoblast and/or osteocyte contribution to the bone phenotype is complicated by simultaneous osteoclast defects in these models. Therefore, we turned to a transgenic mouse model to investigate the direct role of NIK in the osteolineage. Osx‐Cre;NT3 animals (NT3‐Cre +), which bear a constitutively active NIK allele (NT3) driven by Osx‐Cre, were compared with their Cre‐negative, Control (Ctrl) littermates. NT3‐Cre + mice had elevated serum P1NP and CTX levels. Despite this high turnover state, µCT showed that constitutive activation of NIK resulted in a net increase in basal bone mass in both cortical and cancellous compartments. Furthermore, NT3‐Cre + mice exhibited a greater anabolic response following mechanical loading compared with controls. We next performed RNA‐Seq on nonloaded and loaded tibias to elucidate possible mechanisms underlying the increased bone anabolism seen in NT3‐Cre + mice. Hierarchical clustering revealed two main transcriptional programs: one loading‐responsive and the other NT3 transgene‐driven. Gene ontology (GO) analysis indicated a distinct upregulation of receptor, kinase, and growth factor activities including Wnts, as well as a calcium‐response signature in NT3‐Cre + limbs. The promoters of these GO‐term associated genes, including many known to be bone‐anabolic, were highly enriched for multiple κB recognition elements (κB‐RE) relative to the background frequency in the genome. The loading response in NT3‐Cre + mice substantially overlapped (>90%) with Ctrl. Surprisingly, control animals had 10‐fold more DEGs in response to loading. However, most top DEGs shared between genotypes had a high incidence of multiple κB‐RE in their promoters. Therefore, both transcriptional programs (loading‐responsive and NT3 transgene‐driven) are modulated by NF‐κB. Our studies uncover a previously unrecognized role for NF‐κB in the promotion of both basal and mechanically stimulated bone formation. © 2019 American Society for Bone and Mineral Research.

Prolonged effect of zoledronic acid on bone mineral density and turnover in HIV‐infected adults on tenofovir: a randomized, open‐label study

30-07-2019 – A Carr, SJ Kerr, R Richardson, P Ebeling, N Pocock, J Rojas, E Martinez, J Hoy, ZEST study investigators

Journal Article

ABSTRACTZoledronic acid (ZOL) 5 mg annually was more effective than tenofovir disoproxil fumarate (TDF) switching at increasing bone mineral density (BMD) over 24 months in HIV‐infected, osteopenic adults. To determine whether the effects of ZOL would persist without further infusions, we compared changes in left hip and spine BMD over 36 months in participants randomised to ZOL 5 mg at baseline and Month 12 (and to continue TDF) or to switch TDF (without receiving ZOL). We also compared changes in the plasma bone turnover markers (BTMs) C‐terminal telopeptide of type 1 collagen (CTX; bone resorption) and procollagen type 1 N propeptide (P1NP; bone formation) and determined whether CTX and P1NP changes at Month 3 predicted BMD changes at Month 36. Changes were compared in the per‐protocol populations, which included 32 (74%) of 43 participants randomised to ZOL and 37 (88%) of 42 participants who switched TDF. Despite not receiving ZOL after Month 12, mean hip and spine BMD change from baseline were stable and remained greater with ZOL at Month 36 than with TDF switching (spine: 7.5% vs 2.7%, mean difference 4.7%, p < 0.001; hip: 5.5% vs 1.5%, mean difference 4.0%, p < 0.001). CTX and P1NP levels declined in both groups, but significantly more with ZOL. Only percent changes in P1NP at Month 3 correlated inversely with BMD changes at Month 36 (spine, rho = −0.442, p < 0.001; hip, rho = −0.373, p = 0.002). Two infusions of ZOL (in the presence of ongoing TDF) yielded sustained BMD increases through Month 36 that remained greater than with TDF switching.
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Multimodal 18FFDG PET/CT Is a Direct Readout for Inflammatory Bone Repair: A Longitudinal Study in TNFα Transgenic Mice

30-07-2019 – Silvia Hayer, Markus Zeilinger, Volker Weiss, Monika Dumanic, Markus Seibt, Birgit Niederreiter, Tetyana Shvets, Florian Pichler, Wolfgang Wadsak, Bruno K Podesser, Thomas H Helbich, Marcus Hacker, Josef S Smolen, Kurt Redlich, Markus Mitterhauser

Journal Article

ABSTRACTIn rheumatoid arthritis (RA), chronic joint inflammation leading to bone and cartilage damage is the major cause of functional impairment. Whereas reduction of synovitis and blockade of joint damage can be successfully achieved by disease modifying antirheumatic therapies, bone repair upon therapeutic interventions has only been rarely reported. The aim of this study was to use fluorodeoxyglucose (18FFDG) and 18Ffluoride µPET/CT imaging to monitor systemic inflammatory and destructive bone remodeling processes as well as potential bone repair in an established mouse model of chronic inflammatory, erosive polyarthritis. Therefore, human tumor necrosis factor transgenic (h
TNFtg) mice were treated with infliximab, an anti‐TNF antibody, for 4 weeks. Before and after treatment period, mice received either 18FFDG, for detecting inflammatory processes, or 18Ffluoride, for monitoring bone remodeling processes, for PET scans followed by CT scans. Standardized uptake values (SUVmean) were analyzed in various joints and histopathological signs of arthritis, joint damage, and repair were assessed. Longitudinal PET/CT scans revealed a significant decrease in 18FFDG SUVs in affected joints demonstrating complete remission of inflammatory processes due to TNF blockade. In contrast, 18Ffluoride SUVs could not discriminate between different severities of bone damage in h
TNFtg mice. Repeated in vivo CT images proved a structural reversal of preexisting bone erosions after anti‐TNF therapy. Accordingly, histological analysis showed complete resolution of synovial inflammation and healing of bone at sites of former bone erosion. We conclude that in vivo multimodal 18FFDG µPET/CT imaging allows to quantify and monitor inflammation‐mediated bone damage and reveals not only reversal of synovitis but also bone repair upon TNF blockade in experimental arthritis.

Beclin1 Modulates Bone Homeostasis by Regulating Osteoclast and Chondrocyte Differentiation

30-07-2019 – Atsushi Arai, Sol Kim, Vadim Goldshteyn, Terresa Kim, No‐Hee Park, Cun‐Yu Wang, Reuben H. Kim

Journal Article

ABSTRACTAutophagy (ATG), an important cellular recycling process whereby macromolecules or organelles are encapsulated by autophagosome and degraded upon merging with lysosome, has recently been shown to play an essential role in bone biology. However, the involvement of ATG in bone and bone‐related cells remains unclear. Here, we show that Beclin1, an ATG‐related protein involved in ATG initiation, plays a pivotal role in osteoclasts. ATG was activated during osteoclast differentiation in vitro. Beclin1 was enhanced and required for osteoclast differentiation. Mechanistically, we found that TRAF6‐mediated ubiquitination of Beclin1 at K117, but not ULK1‐mediated phosphorylation, is required for RANKL‐stimulated osteoclast differentiation. In vivo, mice lacking Beclin1 in Cst
K‐expressing cells exhibited an increased cortical bone thickness caused by impaired osteoclasts’ function. Interestingly, these mice also exhibited diminished trabecular bone mass, which was associated with a defect in cartilage formation and chondrocyte differentiation. Collectively, our study highlights the functional importance of ATG in osteoclasts and chondrocytes, and identifies ATG as a potential therapeutic target for managing bone‐related diseases. © 2019 American Society for Bone and Mineral Research.

ΔFosB Requires Galanin, but not Leptin, to Increase Bone Mass via the Hypothalamus, but both are needed to increase Energy expenditure

30-07-2019 – Anna Idelevich, Kazusa Sato, Kenichi Nagano, Glenn Rowe, Francesca Gori, Roland Baron

Journal Article

ABSTRACTEnergy metabolism and bone homeostasis share several regulatory pathways. The AP1 transcription factor ΔFos
B and leptin both regulate energy metabolism and bone, yet whether their pathways intersect is not known. Transgenic mice overexpressing ΔFos
B under the control of the Enolase 2 (ENO2) promoter exhibit high bone mass, high energy expenditure, low fat mass, and low circulating leptin levels. Because leptin is a regulator of bone and ΔFos
B acts on leptin‐responsive ventral hypothalamic (VHT) neurons to induce bone anabolism, we hypothesized that regulation of leptin may contribute to the central actions of ΔFos
B in the VHT. To address this question, we used adeno‐associated virus (AAV) expression of ΔFos
B in the VHT of leptin‐deficient ob/ob mice and genetic crossing of ENO2‐ΔFos
B with ob/ob mice. In both models, leptin deficiency prevented ΔFos
B‐triggered reduction in body weight, increase in energy expenditure, increase in glucose utilization, and reduction in pancreatic islet size. In contrast, leptin deficiency failed to prevent ΔFos
B‐triggered increase in bone mass. Unlike leptin deficiency, galanin deficiency blocked both the metabolic and the bone ΔFos
B‐induced effects. Overall, our data demonstrate that, while the catabolic energy metabolism effects of ΔFos
B require intact leptin and galanin signaling, the bone mass–accruing effects of ΔFos
B require galanin but are independent of leptin. © 2019 American Society for Bone and Mineral Research.

Osteogenesis Imperfecta: Skeletal Outcomes After Bisphosphonate Discontinuation at Final Height

29-07-2019 – Marie‐Eve Robinson, Pamela Trejo, Telma Palomo, Francis H Glorieux, Frank Rauch

Journal Article

ABSTRACTIntravenous cyclical bisphosphonates are widely used to treat children with moderate to severe osteogenesis imperfecta (OI). Bisphosphonates are often discontinued when growth is completed, but subsequent skeletal changes have not been studied in detail. We assessed 31 patients (22 females) with OI who had started intravenous bisphosphonates (either pamidronate or zoledronic acid) before 13 years of age, were treated for at least two years (range 4.7–15.7 years) and discontinued treatment after completion of growth, when their age ranged between 13.4–20.0 years (mean: 16.4 years). At 4 years after treatment discontinuation, lumbar spine areal bone mineral density (BMD) had increased by 4% (P < 0.05). Peripheral quantitative computed tomography of the radius showed a decrease in trabecular volumetric BMD at the distal metaphysis of 19% but an increase in cortical volumetric BMD of 4% (P < 0.05 both). None of the patients sustained a new vertebral compression fracture during follow‐up. The proportion of patients with new long‐bone fractures was higher in the two years before treatment discontinuation than in the last two years of follow‐up (42% and 16%, respectively; P < 0.05).
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MiR‐146a deletion protects from bone loss in OVX mice by suppressing RANKL/OPG and M‐CSF in bone microenvironment

29-07-2019 – Jingyu Zhao, Mingjian Huang, Xudong Zhang, Jiajia Xu, Guoli Hu, Xiaoying Zhao, Penglei Cui, Xiaoling Zhang

Journal Article

ABSTRACTMicro
RNAs play important roles in osteoporosis and show great potential for diagnosis and therapy of osteoporosis. Previous studies have demonstrated that mi
R‐146a affects osteoblast (OB) and osteoclast (OC) formation. However, these findings have yet to be identified in vivo, and it is unclear whether mi
R‐146a is related to postmenopausal osteoporosis. Here, we demonstrated that mi
R‐146a knockout protects bone loss in mouse model of estrogen‐deficient osteoporosis, and mi
R‐146a inhibits OB and OC activities in vitro and in vivo. Mi
R‐146a−/− mice displayed the same bone mass as the wild type (WT), but exhibited a stronger bone turnover than the WT did under normal conditions. Nevertheless, mi
R‐146a−/− mice showed an increase in bone mass after undergoing ovariectomy (OVX) in comparison with those subjected to sham operation. OC activities were impaired in the mi
R‐146a−/− mice exposed to estrogen deficiency, which was diametrically opposite to the enhanced bone resorption ability of WT. M‐CSF and RANKL/OPG from a bone microenvironment affect this extraordinary phenomenon. Therefore, our results implicate that mi
R‐146a plays a key role in estrogen deficiency‐induced osteoporosis, and the inhibition of this molecule provides skeleton protection.
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Age‐Related Changes in the Mechanical Regulation of Bone Healing Are Explained by Altered Cellular Mechanoresponse

29-07-2019 – Edoardo Borgiani, Christine Figge, Bettina Kruck, Bettina M Willie, Georg N Duda, Sara Checa

Journal Article

ABSTRACTIncreasing age is associated with a reduced bone regeneration potential and increased risk of morbidities and mortality. A reduced bone formation response to mechanical loading has been shown with aging, and it remains unknown if the interplay between aging and mechanical stimuli during regeneration is similar to adaptation. We used a combined in vivo/in silico approach to investigate age‐related alterations in the mechanical regulation of bone healing and identified the relative impact of altered cellular function on tissue patterns during the regenerative cascade. To modulate the mechanical environment, femoral osteotomies in adult and elderly mice were stabilized using either a rigid or a semirigid external fixator, and the course of healing was evaluated using histomorphometric and micro‐CT analyses at 7, 14, and 21 days post‐surgery. Computer models were developed to investigate the influence of the local mechanical environment within the callus on tissue formation patterns. The models aimed to identify the key processes at the cellular level that alter the mechanical regulation of healing with aging. Fifteen age‐related biological alterations were investigated on two levels (adult and elderly) with a design of experiments setup. We show a reduced response to changes in fixation stability with age, which could be explained by reduced cellular mechanoresponse, simulated as alteration of the ranges of mechanical stimuli driving mesenchymal stem cell differentiation. Cellular mechanoresponse has been so far widely ignored as a therapeutic target in aged patients. Our data hint to mechanotherapeutics as a potential treatment to enhance bone healing in the elderly. © 2019 American Society for Bone and Mineral Research.

Treatment of Autosomal Dominant Hypocalcemia Type 1 With the Calcilytic NPSP795 (SHP635)

26-07-2019 – Mary Scott Roberts, Rachel I Gafni, Beth Brillante, Lori C Guthrie, Jamie Streit, David Gash, Jeff Gelb, Eva Krusinska, Sarah C Brennan, Martin Schepelmann, Daniela Riccardi, Mohd Ezuan Bin Khayat, Donald T Ward, Edward F Nemeth, Ralf Rosskamp, Michael T Collins

Journal Article

ABSTRACTAutosomal dominant hypocalcemia type 1 (ADH1) is a rare form of hypoparathyroidism caused by heterozygous, gain‐of‐function mutations of the calcium‐sensing receptor gene (CAR). Individuals are hypocalcemic with inappropriately low parathyroid hormone (PTH) secretion and relative hypercalciuria. Calcilytics are negative allosteric modulators of the extracellular calcium receptor (Ca
R) and therefore may have therapeutic benefits in ADH1. Five adults with ADH1 due to four distinct CAR mutations received escalating doses of the calcilytic compound NPSP795 (SHP635) on 3 consecutive days. Pharmacokinetics, pharmacodynamics, efficacy, and safety were assessed. Parallel in vitro testing with subject Ca
R mutations assessed the effects of NPSP795 on cytoplasmic calcium concentrations (Ca2+i), and ERK and p38MAPK phosphorylation. These effects were correlated with clinical responses to administration of NPSP795. NPSP795 increased plasma PTH levels in a concentration‐dependent manner up to 129% above baseline (p = 0.013) at the highest exposure levels. Fractional excretion of calcium (FECa) trended down but not significantly so. Blood ionized calcium levels remained stable during NPSP795 infusion despite fasting, no calcitriol supplementation, and little calcium supplementation. NPSP795 was generally safe and well‐tolerated. There was significant variability in response clinically across genotypes. In vitro, all mutant Ca
Rs were half‐maximally activated (EC50) at lower concentrations of extracellular calcium (Ca2+o) compared to wild‐type (WT) Ca
R; NPSP795 exposure increased the EC50 for all Ca
R activity readouts. However, the in vitro responses to NPSP795 did not correlate with any clinical parameters. NPSP795 increased plasma PTH levels in subjects with ADH1 in a dose‐dependent manner, and thus, serves as proof‐of‐concept that calcilytics could be an effective treatment for ADH1. Albeit all mutations appear to be activating at the Ca
R, in vitro observations were not predictive of the in vivo phenotype or the response to calcilytics, suggesting that other parameters impact the response to the drug. © 2019 American Society for Bone and Mineral Research.

Novel Role for Claudin‐11 in the Regulation of Osteoblasts via Modulation of ADAM10‐Mediated Notch Signaling

26-07-2019 – Richard C Lindsey, Weirong Xing, Sheila Pourteymoor, Catrina Godwin, Alexander Gow, Subburaman Mohan

Journal Article

ABSTRACTThe claudin (Cldn) family comprises 27 members of 20 to 34 k
Da transmembrane tight junction proteins. In addition to Cldns’ established canonical role as barriers controlling paracellular flow of molecules, a distinct noncanonical role for them as mediators of cell signaling is now emerging. In our studies evaluating Cldn family expression levels during osteoblast differentiation, Cldn‐11 showed the largest increase (60‐fold). Immunohistochemistry studies revealed high Cldn‐11 expression in trabecular (Tb) bone lining cells. Micro‐CT analysis of femurs and vertebrae of Cldn‐11 knock‐out (KO) mice at 12 weeks of age exhibited a 40% (p < 0.01) reduction in Tb bone volume adjusted for tissue volume compared with control mice, a change caused by significant reductions in Tb number and thickness and increase in Tb separation. Histomorphometry and serum biomarker studies revealed that reduced bone formation, not increased resorption, is the cause for reduced Tb bone volume in the Cldn‐11 KO mice. Cldn‐11 KO osteoblasts expressed reduced ALP and BSP, whereas Cldn‐11 overexpression in MC3T3‐E1 cells increased expression of ALP and BSP. Mechanistically, Cldn‐11 interacted with tetraspanin (Tspan)3 in osteoblasts, and Tspan3 knockdown reduced osteoblast differentiation. Because members of the Tspan family regulate cell functions via Notch signaling, we evaluated whether Cldn‐11/Tspan3 regulates Notch signaling in osteoblasts. Accordingly, Notch targets Hey1 and Hey2 were significantly upregulated in Cldn‐11 overexpressing cultures but downregulated in both Cldn‐11 KO and Tspan3 knockdown osteoblasts. Because ADAM10 has been shown to interact with Tspan family members to regulate Notch signaling, we evaluated whether Cldn‐11 regulates ADAM10 expression. Cldn‐11 overexpressing cells express more mature ADAM10, and an ADAM10 inhibitor blocked the Cldn‐11 effect on osteoblast differentiation. Based on these data, we propose Cldn‐11 as a novel component of an osteoblast cell surface protein complex, comprising Tspan3 and ADAM10, which regulates Notch signaling and cell differentiation. © 2019 American Society for Bone and Mineral Research.

Women With Pregnancy and Lactation–Associated Osteoporosis (PLO) Have Low Bone Remodeling Rates at the Tissue Level

26-07-2019 – Adi Cohen, Mafo Kamanda‐Kosseh, David W Dempster, Hua Zhou, Ralph Müller, Elliott Goff, Ivelisse Colon, Mariana Bucovsky, Julie Stubby, Thomas L Nickolas, Emily M Stein, Robert R Recker, Joan M Lappe, Elizabeth Shane

Journal Article

ABSTRACTPregnancy and lactation–associated osteoporosis (PLO) is a rare, severe, early form of osteoporosis in which young women present with fractures, usually multiple vertebral fractures, during late pregnancy or lactation. In studies of idiopathic osteoporosis (IOP) in premenopausal women, we enrolled 78 women with low‐trauma fractures and 40 healthy controls, all with normal menses and no secondary cause of bone loss. In 15 of the affected women, the PLO subgroup, fractures had occurred during late pregnancy or lactation. We hypothesized that clinical, bone structural, and metabolic characteristics would differ between women with PLO and those with (non‐PLO) IOP and controls. All were evaluated > 12 months postpartum, when structural and remodeling characteristics would be expected to reflect baseline premenopausal status rather than transient postpartum changes. As previously reported, affected subjects (PLO and IOP) had BMD and microarchitectural deficiencies compared to controls. Women with PLO did not differ from those with IOP in terms of age, BMI, body fat, menarcheal age, parity, or age at first pregnancy. However, women with PLO had a more severe clinical presentation than those with IOP: more fractures (5.5 ± 3.3 versus 2.6 ± 2.1; p = 0.005); more vertebral fractures (80% versus 17%; p < 0.001); and higher prevalence of multiple fractures. BMD deficits were more profound and cortical width tended to be lower in PLO. PLO subjects also had significantly lower tissue‐level mineral apposition rate and bone formation rates (0.005 ± 0.005 versus 0.011 ± 0.010 mm2/mm/year; p = 0.006), as well as lower serum P1NP (33 ± 12 versus 44 ± 18 µg/L; p = 0.02) and CTX (257 ± 102 versus 355 ± 193 pg/m
L; p = 0.01) than IOP. The finding that women with PLO have a low bone remodeling state assessed more than a year postpartum increases our understanding of the pathogenic mechanism of PLO. We conclude that women with PLO may have underlying osteoblast functional deficits which could affect their therapeutic response to osteoanabolic medications. © 2019 American Society for Bone and Mineral Research.

Attenuation of NF‐κB in Intestinal Epithelial Cells Is Sufficient to Mitigate the Bone Loss Comorbidity of Experimental Mouse Colitis

25-07-2019 – Ke Ke, Tim (Hung‐Po) Chen, Manoj Arra, Gabriel Mbalaviele, Gaurav Swarnkar, Yousef Abu‐Amer

Journal Article

ABSTRACTSkeletal abnormalities are common comorbidities of inflammatory bowel disease (IBD). Patients suffering from IBD, including ulcerative colitis and Crohns disease, present with skeletal complications. However, the mechanism underpinning IBD‐associated bone loss remains vague. Intestinal inflammation generates an inflammatory milieu at the intestinal epithelium that leads to dysregulation of mucosal immunity through gut‐residing innate lymphoid cells (ILCs) and other cell types. ILCs are recently identified mucosal cells considered as the gatekeeper of gut immunity and their function is regulated by intestinal epithelial cell (IEC)‐secreted cytokines in response to the inflammatory microenvironment. We first demonstrate that serum as well as IECs collected from the intestine of dextran sulfate sodium (DSS)‐induced colitis mice contain high levels of inflammatory and osteoclastogenic cytokines. Mechanistically, heightened inflammatory response of IECs was associated with significant intrinsic activation of NF‐κB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells) in IECs and increased frequency of ILC1, ILC3, and myeloid osteoclast progenitors. Validating the central role of IEC‐specific NF‐κB activation in this phenomenon, conditional expression of constitutively active inhibitor kappa B kinase 2 (IKK2) in IECs in mice recapitulates the majority of the cellular, inflammatory, and osteolytic phenotypes observed in the chemically induced colitis. Furthermore, conditional deletion of IKK2 from IECs significantly attenuated inflammation and bone loss in DSS‐induced colitis. Finally, using the DSS‐induced colitis model, pharmacologic inhibition of IKK2 was effective in reducing frequency of ILC1 and ILC3 cells, attenuated circulating levels of inflammatory cytokines, and halted colitis‐associated bone loss. Our findings identify IKK2 in IECs as viable therapeutic target for colitis‐associated osteopenia.

A Lot of Progress, With More to Be Done: A Response to NIH Pathways to Prevention Report “Research Gaps for Long‐Term Drug Therapies for Osteoporotic Fracture Prevention”

25-07-2019 – Benjamin Z Leder, Bart L Clarke, Elizabeth Shane, Sundeep Khosla, Douglas P Kiel

Journal Article

ABSTRACTThe public health implications of osteoporosis are enormous but the disease remains underdiagnosed and undertreated. In October 2018, the National Institutes of Health (NIH) convened a Pathways to Prevention (P2P) Workshop entitled “Appropriate Use of Drug Therapies for Osteoporotic Fracture Prevention” designed to identify research gaps, suggest future research opportunities, and advance the field through an evidence‐based assessment. By design, the P2P report focused on “gaps” in our knowledge base. Unfortunately, however, the report did not sufficiently acknowledge the current evidence that unequivocally demonstrates the therapeutic efficacy of existing pharmacologic therapies for osteoporosis, which has the potential to exacerbate the current crises in osteoporosis diagnosis and treatment. © 2019 American Society for Bone and Mineral Research.

Pamidronate Administration during Pregnancy and Lactation Induces Temporal Preservation of Maternal Bone Mass in a Mouse Model of Osteogenesis Imperfecta

16-07-2019 – Diana Olvera, Rachel Stolzenfeld, Emily Fisher, Bonnie Nolan, Michelle S. Caird, Kenneth M. Kozloff

Journal Article

Abstract
During pregnancy and lactation, the maternal skeleton undergoes significant bone loss through increased resorption to provide the necessary calcium supply to the developing fetus and suckling neonate. This period of skeletal vulnerability has not been clearly associated with increased maternal fracture risk, but these physiological conditions can exacerbate an underlying metabolic bone condition like osteogenesis imperfecta. While bisphosphonates are commonly used in post‐menopausal women, there are cases where pre‐menopausal women taking bisphosphonates become pregnant. Given bisphosphonates’ long half‐life, there is a need to establish how bisphosphonates affect the maternal skeleton during periods of demanding metabolic bone changes that are critical for the skeletal development of their offspring. In the present study, pamidronate amplified pregnancy‐induced bone mass gains and lactation‐induced bone loss was prevented. This preservation of bone mass was less robust when pamidronate was administered at late stages of lactation compared to early pregnancy and first day of lactation. Pregnancy‐induced osteocyte osteolysis was also observed and was unaffected with pamidronate treatment. No negative skeletal effects were observed in offspring from pamidronate‐treated dams in spite of lactation‐induced bone loss prevention. These findings provide important insight into a treatment window for when pamidronate is most effective in preserving maternal bone mass and provides significant insight to the maternal changes in bone metabolism that maintain calcium homeostasis crucial for fetal and neonatal bone development.
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Association between abdominal aortic calcification, bone mineral density and fracture in older women

16-07-2019 – Joshua R. Lewis, Celeste J. Eggermont, John T. Schousboe, Wai H. Lim, Germaine Wong, Ben Khoo, Marc Sim, MingXiang Yu, Thor Ueland, Jens Bollerslev, Jonathan M Hodgson, Kun Zhu, Kevin E. Wilson, Douglas P. Kiel, Richard L. Prince

Journal Article

ABSTRACTAlthough a relationship between vascular disease and osteoporosis has been recognized, its clinical importance for fracture risk evaluation remains uncertain. Abdominal aortic calcification (AAC), a recognized measure of vascular disease detected on single‐energy images performed for vertebral fracture assessment, may also identify increased osteoporosis risk. In a prospective 10‐year study of 1,024 older predominantly Caucasian women (mean age 75.0±2.6 years) from the Perth Longitudinal Study of Aging cohort we evaluated the association between AAC, skeletal structure and fractures. AAC and spine fracture were assessed at the time of hip densitometry and heel quantitative ultrasound. AAC was scored 0 to 24 (AAC24) and categorized into; low AAC (score 0 and 1, n=459), moderate AAC (score 2‐5, n=373) and severe AAC (score > 6, n=192). Prevalent vertebral fractures were calculated using the Genant semi‐quantitative method. AAC24 scores were inversely related to hip bone mineral density (BMD) (rs=‐0.077, p=0.013) and heel broadband ultrasound attenuation (rs=‐0.074, p=0.020) and stiffness index (rs=‐0.073, p=0.022). In cross‐sectional analyses women with moderate to severe AAC were more likely to have prevalent fracture and LSI detected lumbar spine but not thoracic spine fractures (Mantel‐Haentzel test of trend p < 0.05). For 10‐year incident clinical fractures and fracture‐related hospitalizations women with moderate to severe AAC (AAC24 score >1) had increased fracture risk (HR 1.48 1.15‐1.91, p=0.002; HR 1.46 1.07‐1.99, p=0.019, respectively) compared to women with low AAC. This relationship remained significant after adjusting for age and hip BMD for clinical fractures (HR 1.40 1.08‐1.81, p=0.010) but was attenuated for fracture‐related hospitalizations (HR 1.33 0.98‐1.83, p=0.073). In conclusion, older women with more marked AAC are at higher risk of fracture, not completely captured by bone structural predictors. These findings further support the concept that vascular calcification and bone pathology may share similar mechanisms of causation that remain to be fully elucidated.
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CTLA4‐Ig Directly Inhibits Osteoclastogenesis by Interfering With Intracellular Calcium Oscillations in Bone Marrow Macrophages

16-07-2019 – Hiroyuki Okada, Hiroshi Kajiya, Yasunori Omata, Takumi Matsumoto, Yuiko Sato, Tami Kobayashi, Satoshi Nakamura, Yosuke Kaneko, Shinya Nakamura, Takuma Koyama, Shunichi Sudo, Masashi Shin, Fujio Okamoto, Hisato Watanabe, Naohiro Tachibana, Jun Hirose, Taku Saito, Toshiyuki Takai, Morio Matsumoto, Masaya Nakamura, Koji Okabe, Takeshi Miyamoto, Sakae Tanaka

Journal Article

ABSTRACTCTLA4‐Ig (cytotoxic T‐lymphocyte antigen 4‐immunoglobulin; Abatacept) is a biologic drug for rheumatoid arthritis. CTLA4 binds to the CD80/86 complex of antigen‐presenting cells and blocks the activation of T cells. Although previous reports showed that CTLA4‐Ig directly inhibited osteoclast differentiation, the whole inhibitory mechanism of CTLA4‐Ig for osteoclast differentiation is unclear. Bone marrow macrophages (BMMs) from WT mice were cultured with M‐CSF and RANKL with or without the recombinant mouse chimera CTLA4‐Ig. Intracellular calcium oscillations of BMMs with RANKL were detected by staining with calcium indicator fura‐2 immediately after administration of CTLA4‐Ig or after one day of treatment. Calcium oscillations were analyzed using Fc receptor gamma‐ (Fc
Rγ‐) deficient BMMs. CTLA4‐Ig inhibited osteoclast differentiation and reduced the expression of the nuclear factor of activated T cells NFATc1 in BMMs in vitro. Calcium oscillations in BMMs were suppressed by CTLA4‐Ig both immediately after administration and after one day of treatment. CTLA4‐Ig did not affect osteoclastogenesis and did not cause remarkable changes in calcium oscillations in Fc
Rγ‐deficient BMMs. Finally, to analyze the effect of CTLA4‐Ig in vivo, we used an LPS‐induced osteolysis model. CTLA4‐Ig suppressed LPS‐induced bone resorption in WT mice, not in Fc
Rγ‐deficient mice. In conclusion, CTLA4‐Ig inhibits intracellular calcium oscillations depending on Fc
Rγ and downregulates NFATc1 expression in BMMs. © 2019 American Society for Bone and Mineral Research.

Absence of Dipeptidyl Peptidase 3 increases oxidative stress and causes bone loss

11-07-2019 – Ciro Menale, Lisa J Robinson, Eleonora Palagano, Rosita Rigoni, Marco Erreni, Alejandro J Almarza, Dario Strina, Stefano Mantero, Michela Lizier, Antonella Forlino, Roberta Besio, Marta Monari, Paolo Vezzoni, Barbara Cassani, Harry C Blair, Anna Villa, Cristina Sobacchi

Journal Article

Abstract
Controlling oxidative stress through the activation of antioxidant pathways is crucial in bone homeostasis and impairments of the cellular defense systems involved contribute to the pathogenesis of common skeletal diseases. Here we focused on the dipeptidyl peptidase 3 (DPP3), a poorly investigated ubiquitous zinc‐dependent exopeptidase activating the Keap1‐Nrf2 antioxidant pathway. We demonstrated Dpp3 expression in bone and, to understand its role in this compartment, we generated a Dpp3 knockout (KO) mouse model and specifically investigated the skeletal phenotype. Adult Dpp3 KO mice showed a mild growth defect, a significant increase in bone marrow cellularity and bone loss mainly caused by increased osteoclast activity. Overall, in the mouse model lack of DPP3 resulted in sustained oxidative stress and in alterations of bone microenvironment favoring the osteoclast compared to the osteoblast lineage. Accordingly, in vitro studies revealed that Dpp3 KO osteoclasts had an inherent increased resorptive activity and ROS production, which on the other hand made them prone to apoptosis. Moreover, absence of DPP3 augmented bone loss after estrogen withdrawal in female mice, further supporting its relevance in the framework of bone pathophysiology. Overall, we demonstrated a non‐redundant role for DPP3 in the maintenance of bone homeostasis and proposed that DPP3 might represent a possible new osteoimmunological player and a marker of human bone loss pathology.
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Complementarity of Cohort Studies and Randomized Controlled Trials

11-07-2019 – John A Eisman, Piet Geusens, Joop van den Bergh

Letter

Overestimation of the Limitations of Randomized Controlled Trials

11-07-2019 – Jonathan Bergman, Anna Nordström, Peter Nordström

Letter

Design and Preclinical Development of TransCon PTH, an Investigational Sustained‐Release PTH Replacement Therapy for Hypoparathyroidism

10-07-2019 – Lars Holten‐Andersen, Susanne Pihl, Caroline E. Rasmussen, Joachim Zettler, Guillaume Maitro, Julia Baron, Stefan Heinig, Eric Hoffmann, Thomas Wegge, Mathias Krusch, Frank Faltinger, Steffen Killian, Kennett Sprogoe, David B. Karpf, Vibeke Miller Breinholt, Felix Cleemann

Journal Article

ABSTRACTHypoparathyroidism (HP) is a condition of PTH deficiency leading to abnormal calcium and phosphate metabolism. The mainstay of therapy consists of vitamin D and calcium supplements, as well as adjunct Natpara PTH(1‐84). However, neither therapy optimally controls urinary calcium (u
Ca) or significantly reduces the incidence of hypercalcemia and hypocalcemia. Trans
Con PTH, a sustained‐release prodrug of PTH(1‐34) in development for the treatment of HP, was designed to overcome these limitations. To determine the pharmacokinetics and pharmacodynamics of Trans
Con PTH, single and repeat s.c. dose studies were performed in rats and monkeys. Trans
Con PTH demonstrated a half‐life of 28 and 34 in rats and monkeys, respectively, after repeated dosing, an infusion‐like profile of the released PTH, characterized by low peak‐to‐trough levels, was obtained in both species. In intact rats and monkeys, daily subcutaneous administration of Trans
Con PTH was associated with increases in serum calcium (s
Ca) levels and decreases in serum phosphate levels (s
P). In monkeys, at a single dose of Trans
Con PTH that increased s
Ca levels within the normal range, a concurrent decrease in u
Ca excretion was observed. In 4‐week repeat‐dose studies in intact rats and monkeys, u
Ca excretion was comparable to controls across all dose levels despite increases in s
Ca levels. Further, in a rat model of HP, Trans
Con PTH normalized s
Ca and s
P levels 24 h per day. This was in contrast to only transient trends towards normalization of s
Ca and s
P levels with an up to 6‐fold higher molar dose of PTH(1‐84). Following repeated dosing to HP rats, u
Ca excretion transiently increased corresponding to increases in s
Ca above normal range, but at the end of the treatment period u
Ca excretion was generally comparable to sham controls. Trans
Con PTH was well tolerated and the observed pharmacokinetics and pharmacodynamics were in line with the expected action of physiological replacement of PTH.
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Regulation of the bone vascular network is sexually dimorphic

03-07-2019 – Alice Goring, Aikta Sharma, Behzad Javaheri, Rosanna CG Smith, Janos M Kanczler, Alan Boyde, Eric Hesse, Sumeet Mahajan, Bjorn R Olsen, Andrew A Pitsillides, Philipp Schneider, Richard OC Oreffo, Claire E Clarkin

Journal Article

Abstract
Osteoblast (OB) lineage cells are an important source of vascular endothelial growth factor (VEGF), which is critical for bone growth and repair. During bone development, pubertal differences in males and females exist, but little is known about whether VEGF signalling contributes to skeletal sexual dimorphism. We have found that in mice, conditional disruption of VEGF in osteocalcin expressing cells (Ocn
VEGFKO) exerts a divergent influence on morphological, cellular, and whole bone properties between sexes. Furthermore, we describe an underlying sexual divergence in VEGF signalling in OB cultures in vitro independent of circulating sex‐hormones. High‐resolution synchrotron computed tomography and backscattered scanning electron microscopy revealed, in males, extensive unmineralised osteoid encasing enlarged blood vessel canals and osteocyte lacunae in cortical bone following VEGF deletion, which contributed to increased porosity. VEGF was deleted in male and female long bone‐derived OBs (OBVEGKO) in vitro and Raman spectroscopic analyses of mineral and matrix repertoires highlighted differences between male and female OBVEGFKO cells, with increased immature phosphate species prevalent in male OBVEGFKO cultures versus WT. Further sexual dimorphism was observed in bone marrow endothelial cell gene expression in vitro following VEGF deletion and in sclerostin protein expression, which was increased in male Ocn
VEGFKO bones versus WT. The impact of altered OB matrix composition following VEGF deletion on whole bone geometry was assessed between sexes, although significant differences between Ocn
VEGFKO and WT were identified only in females. Our results suggest that bone‐derived VEGF regulates matrix mineralisation and vascularisation distinctly in males and females which results in divergent physical bone traits.
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Glycemic control and insulin treatment alter fracture risk in older men with type 2 diabetes mellitus

03-07-2019 – Richard H. Lee, Richard Sloane, Carl Pieper, Kenneth W. Lyles, Robert A. Adler, Courtney Houtven, Joanne LaFleur, Cathleen Colón‐Emeric

Journal Article

ABSTRACTDiabetes mellitus among older men has been associated with increased bone mineral density, but paradoxically increased fracture risk. Given the interactions among medication treatment, glycemic control, and diabetes‐associated comorbidities, the relative effects of each factor remains unclear. This retrospective study includes 652,901 male Veterans age ≥65 years with diabetes and baseline hemoglobin A1c (Hb
A1c) value. All subjects received primary care in the Veterans Health Administration (VHA) from 2000 to 2010. Administrative data included ICD9 diagnoses and pharmacy records, and was linked to Medicare fee‐for‐service data. Hazard ratios for any clinical fracture and hip fracture were calculated using competing risk hazards models, adjusted for fracture risk factors including age, race/ethnicity, BMI, alcohol and tobacco use, rheumatoid arthritis, corticosteroid use, as well as diabetes‐related comorbidities including cardiovascular disease, chronic kidney disease, and peripheral neuropathy. Hb
A1c < 6.5% was associated with a higher risk of any clinical fracture HR 1.08 (95%CI: 1.06–1.11), compared to the reference Hb
A1c of 7.5‐8.5%. Fracture risk was not increased among those with A1c ≥ 8.5%, nor among those with A1c 6.5‐7.5%. Use of insulin was independently associated with greater risk of fracture (HR 1.10, 95% CI: 1.07–1.12). There was a significant interaction between insulin use and Hb
A1c level, (P < 0.001), such that those using insulin with Hb
A1c < 6.5% had HR 1.23 and those with Hb
A1c 6.5‐7.5% had HR 1.15. Metformin use was associated with decreased fracture risk (HR 0.88, 95% CI: 0.87–0.90). We conclude that among older men with diabetes, those with Hb
A1c lower than 6.5% are at increased risk for any clinical and hip fracture. Insulin use is associated with higher fracture risk, especially among those with tight glycemic control. Our findings demonstrate the importance of the treatment regimen and avoiding hypoglycemia for fracture prevention in older men with diabetes.
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Treatment of Human Immunodeficiency Virus Infection With Tenofovir Disoproxil Fumarate–Containing Antiretrovirals Maintains Low Bone Formation Rate, But Increases Osteoid Volume on Bone Histomorphometry

03-07-2019 – Janaina Ramalho, Carolina Steller Wagner Martins, Juliana Galvão, Luzia N Furukawa, Wagner V Domingues, Ivone B Oliveira, Luciene M dos Reis, Rosa MR Pereira, Thomas L Nickolas, Michael T Yin, Margareth Eira, Vanda Jorgetti, Rosa MA Moyses

Journal Article

ABSTRACTBone mineral density (BMD) loss is a known complication of human immunodeficiency virus (HIV) infection and its treatment, particularly with tenofovir disoproxil fumarate (TDF)‐containing antiretroviral regimens. Although renal proximal tubular dysfunction and phosphaturia is common with TDF, it is unknown whether BMD loss results from inadequate mineralization. We evaluated change in BMD by dual‐energy X‐ray absorptiometry (DXA) and bone histomorphometry by tetracycline double‐labeled transiliac crest biopsies in young men living with HIV before (n = 20) and 12 months after (n = 16) initiating TDF/lamivudine/efavirenz. We examined relationships between calciotropic hormones, urinary phosphate excretion, pro‐inflammatory and pro‐resorptive cytokines, and bone remodeling‐related proteins with changes in BMD and histomorphometry. Mean age was 29.6 ± 5.5 years, with mean CD4 + T cell count of 473 ± 196 cells/mm3. At baseline, decreased bone formation rate and increased mineralization lag time were identified in 16 (80%) and 12 (60%) patients, respectively. After 12 months, we detected a 2% to 3% decrease in lumbar spine and hip BMD by DXA. By histomorphometry, we observed no change in bone volume/total volume (BV/TV) and trabecular parameters, but rather, increases in cortical thickness, osteoid volume, and osteoblast and osteoclast surfaces. We did not observe significant worsening of renal phosphate excretion or mineralization parameters. Increases in PTH correlated with decreased BMD but not histomorphometric parameters. Overall, these data suggest abnormalities in bone formation and mineralization occur with HIV infection and are evident at early stages. With TDF‐containing antiretroviral therapy (ART), there is an increase in bone remodeling, reflected by increased osteoblast and osteoclast surfaces, but a persistence in mineralization defect, resulting in increased osteoid volume. © 2019 American Society for Bone and Mineral Research

Relationship Between Sex Steroids and Deterioration of Bone Microarchitecture in Older Men: The Prospective STRAMBO Study

27-06-2019 – Anne Piot, Roland D Chapurlat, Bruno Claustrat, Pawel Szulc

Journal Article

ABSTRACTIn older men, low estrogen levels are associated with poor bone microarchitecture. Data on androgens are discordant. We studied the link between baseline sex steroid levels (total 17β ‐estradiol 17βE2, total testosterone t
T, calculated bioavailable 17βE2 bio‐17βE2, and apparent free testosterone concentration AFTC) and bone microarchitecture deterioration assessed prospectively in a 820 older men followed for 8 years. Bone microarchitecture was assessed by HR‐p
QCT at baseline, then after 4 and 8 years. At both the skeletal sites, the bone microarchitecture deterioration rate did not correlate with serum levels of t
T and 17βE2. At the distal radius, cortical area (Ct.
Ar) decreased more rapidly in the lowest versus the highest AFTC quartile. At the distal tibia, cortical thickness (Ct.
Th) decreased and trabecular area (Tb.
Ar) increased more rapidly in the highest versus the lowest AFTC quartile. At the tibia, bone mineral content (BMC), total volumetric bone mineral density (Tt.v
BMD), Ct.
Th, and Ct.
Ar decreased, whereas Tb.
Ar increased faster in the lowest versus the highest bio‐17βE2 quartile. In men who had both AFTC and bio‐17βE2 in the lowest quartile (high‐risk group), distal radius cortical v
BMD (Ct.v
BMD) decreased more rapidly compared with men who had both hormones in the three upper quartiles (reference group). At the distal tibia, Tt.v
BMD, Ct.
Th, Ct.
Ar, and Ct.v
BMD decreased, whereas Tb.
Ar increased more rapidly in the high‐risk group versus the reference group. In men receiving androgen deprivation therapy (ADT) for prostate cancer, BMC, Tt.v
BMD, Ct.
Th, Ct.
Ar, and Ct.v
BMD decreased, whereas Tb.
Ar increased more rapidly than in men not receiving ADT at both the skeletal sites. Thus, in older men followed up prospectively, low levels of bio‐17βE2, and to a smaller extent AFTC, are associated with accelerated cortical bone deterioration. Cortical bone deterioration was strongly accelerated in men receiving ADT who had very low levels of all sex steroids.

Rmrp mutation disrupts chondrogenesis and bone ossification in zebrafish model of cartilage‐hair hypoplasia via enhanced Wnt/β‐catenin signaling

25-06-2019 – Xianding Sun, Ruobin Zhang, Mi Liu, Hangang Chen, Liang Chen, Fengtao Luo, Dali Zhang, Junlan Huang, Fangfang Li, Zhenhong Ni, Huabing Qi, Nan Su, Min Jin, Jing Yang, Qiaoyan Tan, Xiaolan Du, Bo Chen, Haiyang Huang, Shuai Chen, Liangjun Yin, Xiaoling Xu, Chuxia Deng, Lingfei Luo, Yangli Xie, Lin Chen

Journal Article

ABSTRACTCartilage‐hair hypoplasia (CHH) is an autosomal recessive metaphyseal chondrodysplasia characterized by bone dysplasia and many other highly variable features. The responsible gene for CHH has been identified to be the RNA component of the mitochondrial RNA processing endoribonuclease (RMRP) gene. Currently, the pathogenesis of osteochondrodysplasia and extraskeletal manifestations in CHH patients remains incompletely understood, furthermore, there are no viable animal models for CHH. We generated a rmrp knockout zebrafish model to study the developmental mechanisms of CHH. We found that rmrp is required for the patterning and shaping of pharyngeal arches. Rmrp mutation inhibits the intramembranous ossification of skull bones and promotes vertebrae ossification. The abnormalities of endochondral bone ossification are variable depending on the degree of dysregulated chondrogenesis. Moreover, rmrp mutation inhibits cell proliferation and promotes apoptosis through dysregulating the expressions of cell cycle and apoptosis related genes. Furthermore, we demonstrate that rmrp mutation up‐regulates canonical Wnt/β‐catenin signaling and pharmacological inhibition of Wnt/β‐catenin could partially alleviate the chondrodysplasia and increased vertebrae mineralization in rmrp mutants. Our study, by establishing a novel zebrafish model for CHH, partially reveals the underlying mechanism of CHH, which also deepen our understanding of the role of rmrp in skeleton development.
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Loss of the Vitamin D Receptor in Human Breast Cancer Cells Promotes Epithelial to Mesenchymal Cell Transition and Skeletal Colonization

25-06-2019 – Konstantin Horas, Yu Zheng, Colette Fong‐Yee, Eugenie Macfarlane, Jeline Manibo, Yunzhao Chen, Jeremy Qiao, Mingxuan Gao, Nancy Haydar, Michelle M McDonald, Peter I Croucher, Hong Zhou, Markus J Seibel

Journal Article

ABSTRACTExpression of the vitamin D receptor (VDR) is thought to be associated with neoplastic progression. However, the role of the VDR in breast cancer metastasis to bone and the molecular mechanisms underlying this process are unknown. Employing a rodent model (female Balb/c nu/nu mice) of systemic metastasis, we here demonstrate that knockdown of the VDR strongly increases the metastatic potential of MDA‐MB‐231 human breast cancer cells to bone, resulting in significantly greater skeletal tumor burden. Ablation of VDR expression promotes cancer cell mobility (migration) and invasiveness, thereby facilitating skeletal colonization. Mechanistically, these changes in tumor cell behavior are attributable to shifts in the expression of proteins involved in cell adhesion, proliferation, and cytoskeletal organization, patterns characteristic for epithelial‐to‐mesenchymal cell transition (EMT). In keeping with these experimental findings, analyses of human breast cancer specimens corroborated the association between VDR expression, EMT‐typical changes in protein expression patterns, and clinical prognosis. Loss of the VDR in human breast cancer cells marks a critical point in oncogenesis by inducing EMT, promoting the dissemination of cancer cells, and facilitating the formation of tumor colonies in bone. © 2019 American Society for Bone and Mineral Research.

Combined Collagen‐Induced Arthritis and Organic Dust‐Induced Airway Inflammation to Model Inflammatory Lung Disease in Rheumatoid Arthritis

24-06-2019 – Jill A Poole, Geoffrey M Thiele, Katherine Janike, Amy J Nelson, Michael J Duryee, Kathryn Rentfro, Bryant R England, Debra J Romberger, Joseph M Carrington, Dong Wang, Benjamin J Swanson, Lynell W Klassen, Ted R Mikuls

Journal Article

ABSTRACTRheumatoid arthritis (RA) is characterized by extra‐articular involvement including lung disease, yet the mechanisms linking the two conditions are poorly understood. The collagen‐induced arthritis (CIA) model was combined with the organic dust extract (ODE) airway inflammatory model to assess bone/joint–lung inflammatory outcomes. DBA/1J mice were intranasally treated with saline or ODE daily for 5 weeks. CIA was induced on days 1 and 21. Treatment groups included sham (saline injection/saline inhalation), CIA (CIA/saline), ODE (saline/ODE), and CIA +  ODE (CIA/ODE). Arthritis inflammatory scores, bones, bronchoalveolar lavage fluid, lung tissues, and serum were assessed. In DBA/1J male mice, arthritis was increased in CIA +  ODE >  CIA >  ODE versus sham. Micro‐computed tomography (µCT) demonstrated that loss of BMD and volume and deterioration of bone microarchitecture was greatest in CIA +  ODE. However, ODE‐induced airway neutrophil influx and inflammatory cytokine/chemokine levels in lavage fluids were increased in ODE >  CIA +  ODE versus sham. Activated lung CD11c+CD11b+ macrophages were increased in ODE >  CIA +  ODE >  CIA pattern, whereas lung hyaluronan, fibronectin, and amphiregulin levels were greatest in CIA +  ODE. Serum autoantibody and inflammatory marker concentrations varied among experimental groups. Compared with male mice, female mice showed less articular and pulmonary disease. The interaction of inhalation‐induced airway inflammation and arthritis induction resulted in compartmentalized responses with the greatest degree of arthritis and bone loss in male mice with combined exposures. Data also support suppression of the lung inflammatory response, but increases in extracellular matrix protein deposition/interstitial disease in the setting of arthritis. This coexposure model could be exploited to better understand and treat RA–lung disease.

Bone‐Forming and Antiresorptive Effects of Romosozumab in Postmenopausal Women With Osteoporosis: Bone Histomorphometry and Microcomputed Tomography Analysis After 2 and 12 Months of Treatment

24-06-2019 – Pascale Chavassieux, Roland Chapurlat, Nathalie Portero‐Muzy, Jean‐Paul Roux, Pedro Garcia, Jacques P Brown, Cesar Libanati, Rogely W Boyce, Andrea Wang, Andreas Grauer

Journal Article

ABSTRACTSclerostin, a protein produced by osteocytes, inhibits bone formation. Administration of sclerostin antibody results in increased bone formation in multiple animal models. Romosozumab, a humanized sclerostin antibody, has a dual effect on bone, transiently increasing serum biochemical markers of bone formation and decreasing serum markers of bone resorption, leading to increased BMD and reduction in fracture risk in humans. We aimed to evaluate the effects of romosozumab on bone tissue. In a subset of 107 postmenopausal women with osteoporosis in the multicenter, international, randomized, double‐blind, placebo‐controlled Fracture Study in Postmenopausal Women with Osteoporosis (FRAME), transiliac bone biopsies were performed either after 2 (n = 34) or 12 (n = 73) months of treatment with 210 mg once monthly of romosozumab or placebo to evaluate histomorphometry and microcomputed tomography‐based microarchitectural endpoints. After 2 months, compared with either baseline values assessed after a quadruple fluorochrome labeling or placebo, significant increases (P < 0.05 to P < 0.001) in dynamic parameters of formation (median MS/BS: romosozumab 1.51% and 5.64%; placebo 1.60% and 2.31% at baseline and month 2, respectively) were associated with a significant decrease compared with placebo in parameters of resorption in cancellous (median ES/BS: placebo 3.4%, romosozumab 1.8%; P = 0.022) and endocortical (median ES/BS: placebo 6.3%, romosozumab 1.6%; P = 0.003) bone. At 12 months, cancellous bone formation was significantly lower (P < 0.05 to P < 0.001) in romosozumab versus placebo and the lower values for resorption endpoints seen at month 2 persisted (P < 0.001), signaling a decrease in bone turnover (P = 0.006). No significant change was observed in periosteal and endocortical bone. This resulted in an increase in bone mass and trabecular thickness with improved trabecular connectivity, without significant modification of cortical porosity at month 12. In conclusion, romosozumab produced an early and transient increase in bone formation, but a persistent decrease in bone resorption. Antiresorptive action eventually resulted in decreased bone turnover. This effect resulted in significant increases in bone mass and improved microarchitecture.

Ultrasound‐Based Estimates of Cortical Bone Thickness and Porosity Are Associated With Nontraumatic Fractures in Postmenopausal Women: A Pilot Study

19-06-2019 – J‐G Minonzio, N Bochud, Q Vallet, D Ramiandrisoa, A Etcheto, K Briot, S Kolta, C Roux, P Laugier

Journal Article

ABSTRACTRecent ultrasound (US) axial transmission techniques exploit the multimode waveguide response of long bones to yield estimates of cortical bone structure characteristics. This pilot cross‐sectional study aimed to evaluate the performance at the one‐third distal radius of a bidirectional axial transmission technique (BDAT) to discriminate between fractured and nonfractured postmenopausal women. Cortical thickness (Ct.
Th) and porosity (Ct.
Po) estimates were obtained for 201 postmenopausal women: 109 were nonfractured (62.6 ± 7.8 years), 92 with one or more nontraumatic fractures (68.8 ± 9.2 years), 17 with hip fractures (66.1 ± 10.3 years), 32 with vertebral fractures (72.4 ± 7.9 years), and 17 with wrist fractures (67.8 ± 9.6 years). The areal bone mineral density (a
BMD) was obtained using DXA at the femur and spine. Femoral a
BMD correlated weakly, but significantly with Ct.
Th (R = 0.23, p < 0.001) and Ct.
Po (R = ‐0.15, p < 0.05). Femoral a
BMD and both US parameters were significantly different between the subgroup of all nontraumatic fractures combined and the control group (p < 0.05). The main findings were that (1) Ct.
Po was discriminant for all nontraumatic fractures combined (OR = 1.39; area under the receiver operating characteristic curve AUC equal to 0.71), for vertebral (OR = 1.96; AUC = 0.84) and wrist fractures (OR = 1.80; AUC = 0.71), whereas Ct.
Th was discriminant for hip fractures only (OR = 2.01; AUC = 0.72); there was a significant association (2) between increased Ct.
Po and vertebral and wrist fractures when these fractures were not associated with any measured a
BMD variables; (3) between increased Ct.
Po and all nontraumatic fractures combined independently of a
BMD neck; and (4) between decreased Ct.
Th and hip fractures independently of a
BMD femur. BDAT variables showed comparable performance to that of a
BMD neck with all types of fractures (OR = 1.48; AUC = 0.72) and that of a
BMD femur with hip fractures (OR = 2.21; AUC = 0.70). If these results are confirmed in prospective studies, cortical BDAT measurements may be considered useful for assessing fracture risk in postmenopausal women.

Osteocyte Death and Bone Overgrowth in Mice Lacking Fibroblast Growth Factor Receptors 1 and 2 in Mature Osteoblasts and Osteocytes

17-06-2019 – Jennifer McKenzie, Craig Smith, Kannan Karuppaiah, Joshua Langberg, Matthew J Silva, David M Ornitz

Journal Article

ABSTRACTFibroblast growth factor (FGF) signaling pathways have well‐established roles in skeletal development, with essential functions in both chondrogenesis and osteogenesis. In mice, previous conditional knockout studies suggested distinct roles for FGF receptor 1 (FGFR1) signaling at different stages of osteogenesis and a role for FGFR2 in osteoblast maturation. However, the potential for redundancy among FGFRs and the mechanisms and consequences of stage‐specific osteoblast lineage regulation were not addressed. Here, we conditionally inactivate Fgfr1 and Fgfr2 in mature osteoblasts with an Osteocalcin (OC)‐Cre or Dentin matrix protein 1 (Dmp1)‐Cre
ER driver. We find that young mice lacking both receptors or only FGFR1 are phenotypically normal. However, between 6 and 12 weeks of age, OC‐Cre Fgfr1/Fgfr2 double‐ and Fgfr1 single‐conditional knockout mice develop a high bone mass phenotype with increased periosteal apposition, increased and disorganized endocortical bone with increased porosity, and biomechanical properties that reflect increased bone mass but impaired material properties. Histopathological and gene expression analyses show that this phenotype is preceded by a striking loss of osteocytes and accompanied by activation of the Wnt/β‐catenin signaling pathway. These data identify a role for FGFR1 signaling in mature osteoblasts/osteocytes that is directly or indirectly required for osteocyte survival and regulation of bone mass during postnatal bone growth.

Compromised Exercise Capacity and Mitochondrial Dysfunction in the Osteogenesis Imperfecta Murine (oim) Mouse Model

13-06-2019 – Victoria L Gremminger, Youngjae Jeong, Rory P Cunningham, Grace M Meers, R Scott Rector, Charlotte L Phillips

Journal Article

ABSTRACTOsteogenesis imperfecta (OI) is a heritable connective tissue disorder that most often arises from type I collagen—COL1A1 and COL1A2—gene defects leading to skeletal fragility, short stature, blue‐gray sclera, and muscle weakness. Relative to the skeletal fragility, muscle weakness is much less understood. Recent investigations into OI muscle weakness in both patients and mouse models have revealed the presence of an inherent muscle pathology. Understanding the mechanisms responsible for OI muscle weakness is critical, particularly in light of the extensive cross‐talk between muscle and bone via mechanotransduction and biochemical signaling. In the following study we initially subjected WT and oim/oim mice, modeling severe human OI type III, to either weight‐bearing (voluntary wheel‐running) or non‐weight‐bearing (swimming) exercise regimens as a modality to improve muscle strength and ultimately bone strength. The oim/oim mice ran only 35% to 42% of the distance run by age‐ and sex‐matched WT mice and exhibited little improvement with either exercise regimen. Upon further investigation, we determined that oim/oim gastrocnemius muscle exhibited severe mitochondrial dysfunction as characterized by a 52% to 65% decrease in mitochondrial respiration rates, alterations in markers of mitochondrial biogenesis, mitophagy, and the electron transport chain components, as well as decreased mitochondrial citrate synthase activity, relative to age‐ and sex‐matched WT gastrocnemius muscle. Thus, mitochondrial dysfunction in the oim/oim mouse likely contributes to compromised muscle function and reduced physical activity levels.

Chondrocyte‐Specific RUNX2 Overexpression Accelerates Post‐traumatic Osteoarthritis Progression in Adult Mice

12-06-2019 – Sarah E Catheline, Donna Hoak, Martin Chang, John P Ketz, Matthew J Hilton, Michael J Zuscik, Jennifer H Jonason

Journal Article

ABSTRACTRUNX2 is a transcription factor critical for chondrocyte maturation and normal endochondral bone formation. It promotes the expression of factors catabolic to the cartilage extracellular matrix and is upregulated in human osteoarthritic cartilage and in murine articular cartilage following joint injury. To date, in vivo studies of RUNX2 overexpression in cartilage have been limited to forced expression in osteochondroprogenitor cells preventing investigation into the effects of chondrocyte‐specific RUNX2 overexpression in postnatal articular cartilage. Here, we used the Rosa26 Runx2 allele in combination with the inducible Col2a1 Cre
ERT2 transgene or the inducible Acan Cre
ERT2 knock‐in allele to achieve chondrocyte‐specific RUNX2 overexpression (OE) during embryonic development or in the articular cartilage of adult mice, respectively. RUNX2 OE was induced at embryonic day 13.5 (E13.5) for all developmental studies. Histology and in situ hybridization analyses suggest an early onset of chondrocyte hypertrophy and accelerated terminal maturation in the limbs of the RUNX2 OE embryos compared to control embryos. For all postnatal studies, RUNX2 OE was induced at 2 months of age. Surprisingly, no histopathological signs of cartilage degeneration were observed even 6 months following induction of RUNX2 OE. Using the meniscal/ligamentous injury (MLI), a surgical model of knee joint destabilization and meniscal injury, however, we found that RUNX2 OE accelerates the progression of cartilage degeneration following joint trauma. One month following MLI, the numbers of MMP13‐positive and TUNEL‐positive chondrocytes were significantly greater in the articular cartilage of the RUNX2 OE joints compared to control joints and 2 months following MLI, histomorphometry and Osteoarthritis Research Society International (OARSI) scoring revealed decreased cartilage area in the RUNX2 OE joints. Collectively, these results suggest that although RUNX2 overexpression alone may not be sufficient to initiate the OA degenerative process, it may predetermine the rate of OA onset and/or progression following traumatic joint injury.

18F‐NnaF PET/CT IMAGING IN FIBROUS DYSPLASIA OF BONE

22-05-2019 – GeorgiosZ Papadakis, GeorgiosC Manikis, ApostolosH Karantanas, Pablo Florenzano, Ulas Bagci, Kostas Marias, MichaelT Collins, AlisonM Boyce

Journal Article

ABSTRACTFibrous dysplasia (FD) is a mosaic skeletal disorder resulting in fractures, deformity, and functional impairment. Clinical evaluation has been limited by a lack of surrogate endpoints capable of quantitating disease activity. The purpose of this study was to investigate the utility of 18F‐Na
F PET/CT imaging in quantifying disease activity in patients with FD. Fifteen consecutively evaluated subjects underwent whole‐body 18F‐Na
F PET/CT scans, and FD burden was assessed by quantifying FD‐related 18F‐Na
F activity. 18F‐Na
F PET/CT parameters obtained included (i) SUVmax (standardized uptake value SUV of the FD lesion with the highest uptake); (ii) SUVmean (average SUV of all 18F‐Na
F–positive FD lesions); (iii) total volume of all 18F‐Na
F–positive FD lesions (TV); and (iv) total FD lesion activity determined as the product of TV multiplied by SUVmean (TA =  TV ×  SUVmean) (TA). Skeletal outcomes, functional outcomes, and bone turnover markers were correlated with 18F‐Na
F PET/CT parameters. TV and TA of extracranial FD lesions correlated strongly with skeletal outcomes including fractures and surgeries (p values ≤ 0.003). Subjects with impaired ambulation and scoliosis had significantly higher TV and TA values (P < 0.05), obtained from extracranial and spinal lesions, respectively. Craniofacial surgeries correlated with TV and TA of skull FD lesions (P < 0.001). Bone turnover markers, including alkaline phosphatase, N‐telopeptides, and osteocalcin, were strongly correlated with TV and TA (P < 0.05) extracted from FD lesions in the entire skeleton. No associations were identified with SUVmax or SUVmean. Bone pain and age did not correlate with 18F‐Na
F PET/CT parameters. FD burden evaluated by 18F‐Na
F‐PET/CT facilitates accurate assessment of FD activity, and correlates quantitatively with clinically‐relevant skeletal outcomes.

Glucocorticoids Decrease Longitudinal Bone Growth in Paediatric Kidney Transplant Recipients by Stimulating the FGF23/FGFR3 Signalling Pathway

17-05-2019 – Ángela Delucchi, Luis Toro, Rodrigo Alzamora, Victor Barrientos, Magdalena González, Rodrigo Andaur, Pablo León, Francisco Villanueva, Mario Galindo, Facundo Las Heras, Martín Montecino, Daniel Moena, Andrea Lazcano, Viola Pinto, Paulina Salas, María Loreto Reyes, Verónica Mericq, Luis Michea

Journal Article

ABSTRACTRenal transplantation (RTx) is an effective therapy to improve clinical outcomes in paediatric patients with terminal chronic kidney disease. However, chronic immunosuppression with glucocorticoids (GC) reduces bone growth and mineral density. The mechanisms causing GC‐induced growth impairment have not been fully clarified. Fibroblast growth factor 23 (FGF23) is a peptide hormone that regulates phosphate homeostasis and bone growth. In pathological conditions, FGF23 excess or abnormal FGF receptors (FGFR) activity leads to bone growth impairment. Experimental data indicates that FGF23 expression is induced by chronic GC exposure. Therefore, we hypothesize that GC impairs bone growth by increasing FGF23 expression, which has direct effects on bone growth plate. In a post‐hoc analysis of a multicentric randomized clinical trial of prepubertal RTx children treated with early GC withdrawal or chronic GC treatment, we observed that GC withdrawal was associated with improvement in longitudinal growth and bone mineral density, and lower plasma FGF23 levels as compared to chronic GC group. In prepubertal rats, GC‐induced bone growth retardation correlated with increased plasma FGF23 and bone FGF23 expression. Additionally, GC treatment decreased FGFR1 expression while increased FGFR3 expression in mouse tibiae explants. Blockage of FGF23 receptors using a pan‐FGFR antagonist (PD173074), C‐terminal FGF23 (FGF23180‐205) which blocks the binding of FGF23 with FGFR‐Klotho complex or a specific FGFR3 antagonist (P3), prevented GC‐induced growth impairment in vitro. Finally, local administration of PD173074 into tibiae growth plate of GC‐treated rats ameliorated cartilage growth impairment. These results show that GC treatment partially reduces longitudinal bone growth via upregulation of FGF23 and FGFR3 expression, thus suggesting that the FGF23/Klotho/FGFR3 axis at the growth plate could be a potential therapeutic target for the management of GC‐induced growth impairment in children.
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The prevalence of osteoporosis in China, a nationwide, multicenter DXA survey

08-05-2019 – Qiang Zeng, Na Li, Qianqian Wang, Jian Feng, Dongmao Sun, Qiu Zhang, Jiyuan Huang, Qingxiang Wen, Rong Hu, Liang Wang, Yuanzheng Ma, Xiaoxia Fu, Shengyong Dong, Xiaoguang Cheng

Journal Article

Abstract
A number of studies investigated the distribution of BMD values and the prevalence of osteoporosis in China, but their findings varied. Until now, a BMD reference database based on uniform measurements in a large‐scale Chinese population has been lacking. A total of 75,321 Chinese adults aged 20 years and older were recruited from 7 centers between 2008 and 2018. BMD values at the lumbar spine 1‐4, femoral neck, and total femur were measured by GE Lunar dual energy X‐ray absorptiometry systems. BMD values measured in each center were cross‐calibrated by regression equations that were generated by scanning the same European spine phantom 10 times at every center. Cubic and multivariate linear regression were performed to assess associations between BMD values and demographic variables. Sex‐specific prevalence of osteoporosis was age‐standardized based on the year 2010 national census data for the Chinese population. The sex‐specific BMD values at each site were negatively associated with age, positively associated with body mass index levels, and lower in the participants from Southwest China than in those from other geographic regions after multivariate adjustment. Furthermore, BMD values at the femoral neck and total femur decreased with the year of BMD measurement. The peak BMD values at the lumbar spine, femoral neck, and total femur were 1.088 g/cm2, 0.966 g/cm2, and 0.973 g/cm2, respectively, for men and 1.114 g/cm2, 0.843 g/cm2, and 0.884 g/cm2, respectively, for women. The age‐standardized prevalence of osteoporosis at the spine or hip was 6.46% and 29.13% for men and women aged 50 years and older, respectively. Currently a total of 10.9 million men and 49.3 million women in China are estimated to have osteoporosis. In our national examination of BMD, we found that BMD values differed by demographic characteristics. We estimated the age‐standardize prevalence of osteoporosis in China to be 6.46% and 29.13% for men and women aged 50 years and older.
This article is protected by copyright. All rights reserved.