bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced Incidence of Hip Fracture in the Old Order Amish
Authors: Streeten Elizabeth A , Mcbride Daniel J , Lodge Amy L , Pollin Toni I , Stinchcomb David G , Agarwala Richa , Schäffer Alejandro A , Shapiro Jay R , Shuldiner Alan R , Mitchell Braxton D
Published: April 2006

The incidence of hip fracture was estimated in a community of Old Order Amish and compared with available data from non-Amish whites. Hip fracture rates were 40% lower in the Amish, and the Amish also experienced higher BMD.

Introduction: Understanding the patterns of fracture risk across populations could reveal insights about bone health and lead to the earlier detection and prevention of osteoporosis. Toward this aim, we compared hip fracture incidence and bone mineral density (BMD) between an Old Order Amish (OOA) community, characterized by a rural and relatively active lifestyle, and non-Amish U.S. whites.

Materials and Methods: All hospital admissions for hip fracture among OOA individuals in Lancaster County, PA, were identified between 1995 and 1998 from four area hospitals. Hip fracture incidence was calculated by cross-referencing an available Anabaptist genealogy database with communities located within these hospital service areas and compared with non-Amish whites obtained from National Hospital Discharge data. Additionally, BMD at the hip was compared between 287 Amish subjects and non-Amish whites from the National Health and Nutrition Examination Survey III survey.

Results and Conclusions: OOA experienced 42% fewer hip fractures than would be expected had they experienced the same rate of hip fracture as observed in non-Amish whites (p < 0.01) and a higher mean BMD that was significant in women (p < 0.05) but not men. Further evaluation of lifestyle and/or genetic differences between Amish and non-Amish populations may shed insights into etiologic factors influencing hip fracture risk.
Dietary Silicon Intake Is Positively Associated With Bone Mineral Density in Men and Premenopausal Women of the Framingham Offspring Cohort
Authors: Jugdaohsingh Ravin , Tucker Katherine L , Qiao Ning , Cupples L Adrienne , Kiel Douglas P , Powell Jonathan J
Published: April 2006

The role of dietary silicon in bone health in humans is not known. In a cross-sectional, population-based study (2847 participants), associations between dietary silicon intake and BMD were investigated. Dietary silicon correlated positively and significantly with BMD at all hip sites in men and premenopausal women, but not in postmenopausal women, suggesting that increased silicon intake is associated with increased cortical BMD in these populations.

Introduction: Osteoporosis is a burgeoning health and economic issue. Agents that promote bone formation are widely sought. Animal and cellular data suggest that the orthosilicate anion (i.e., dietary silicon) is involved in bone formation. The intake of silicon (Si, ∼30 mg/day) is among the highest for trace elements in humans, but its contribution to bone health is not known.

Materials and Methods: In a cross-sectional, population-based study, we examined the association between silicon intake and bone mineral density (BMD) in 1251 men and 1596 pre- and postmenopausal women in the Framingham Offspring cohort (age, 30–87 years) at four hip sites and lumbar spine, adjusting for all potential confounding factors known to influence BMD and nutrient intake.

Results: Silicon intake correlated positively with adjusted BMD at four hip sites in men and premenopausal women, but not in postmenopausal women. No significant association was observed at the lumbar spine in any group. Categorical analysis by Si intake, or energy-adjusted Si intake, supported these findings, and showed large differences in BMD (up to 10%) between the highest (>40 mg Si/day) and lowest (<14 mg Si/day) quintiles of silicon intake. A significant association at the lumbar spine in men was also observed. Further analyses indicated that some of the effects seen for moderate consumption of alcoholic beverages on BMD might be attributed to Si intake.

Conclusions: These findings suggest that higher dietary silicon intake in men and younger women may have salutary effects on skeletal health, especially cortical bone health, that has not been previously recognized. Confirmation of these results is being sought in a longitudinal study and by assessment of the influence of silicon intake on bone markers in this cohort.
Water Content Measured by Proton-Deuteron Exchange NMR Predicts Bone Mineral Density and Mechanical Properties
Authors: Fernández-Seara Maria A , Wehrli Suzanne L , Takahashi Masaya , Wehrli Felix W
Published: April 2006

NMR was used to measure matrix water content in normal and hypomineralized cortical bone. Water content showed an inverse relationship with mineral content, suggesting it could serve as a surrogate measure for the bone's degree of mineralization.

Introduction: So far, true bone mineral density (DMB; degree of mineralization of bone) can not be measured nondestructively.

Materials and Methods: Here, a new technique combining1H nuclear magnetic resonance (NMR) spectroscopy and deuterium isotope exchange was used to measure water content in cortical bone from two groups of rabbits: a control group and a group fed a low-phosphorus (P) diet to induce hypomineralization of the bone matrix.

Results: NMR-derived water content was higher in the P-depleted group and showed an inverse relationship with mineral content (measured gravimetrically and by31P NMR). Hypomineralized bone was found to be weaker than normal bone as demonstrated by mechanical testing. More importantly, the data showed a strong inverse correlation between water content and bone mechanical properties, which indicates that water content could be predictive of the bone's mechanical competence.

Conclusions: Water content could potentially serve as a surrogate measure for the bone's degree of mineralization, and this technique could be used to study other disorders of mineral homeostasis known to alter the mineralization state of the matrix. Although the method presented here is not suitable for in vivo measurements of bone water content, the authors have previously shown that1H NMR images of bone can be acquired; thus, noninvasive quantification of bone water may be feasible.
Relationship Between Changes in Bone Mineral Density and Fracture Risk Reduction With Antiresorptive Drugs: Some Issues With Meta-Analyses
Authors: Delmas PD , Li Zhengqing , Cooper Cyrus
Published: April 2006

Published meta-analyses have investigated the relationship between changes in BMD and fracture risk reduction observed with antiresorptive agents, with inconsistent results. Many factors may affect the outcome of such analyses. Our work explores some of these factors and illustrates the need for caution in interpreting the results of meta-analyses.

Introduction: The role of the increase in bone mineral density (BMD) in fracture risk reduction observed in osteoporotic patients treated with antiresorptive drugs is unclear. We examined the effects of study selection, the use of summary statistics or individual patient data (IPD) as the basis for the analyses, and the choice of BMD values used on the outcome of meta-analyses.

Materials and Methods: To evaluate the effects of study selection, we performed Poisson regression analyses using the results from a number of published studies. To evaluate the effects of using individual patient data instead of summary statistics, we simulated the IPD for vertebral fracture to match the summary statistics for published trials and compared these results with those based on meta-regression using summary statistics. We also evaluated the effect of varying the BMD increase with treatment (3–8%) used in predicting the fracture risk reductions in these simulations.

Results: The Poisson regression, which found a statistically significant relationship between nonvertebral fracture risk and spinal BMD when 18 trials of varying designs, duration, and sample size were included in the analysis (p = 0.02), was no longer significant when the analysis was based on the 7 large studies that were placebo-controlled, at least 3 years in duration (at least 1000 patient-years). Meta-analyses of simulated IPD from 12 trials of six antiresorptive agents gave accurate results regardless of the proportion of vertebral risk reduction assumed to be related to BMD change, whereas meta-regression based on summary statistics always produced an estimate around 50%. When the actual data from two risedronate studies were analyzed, the meta-regression based on summary statistics demonstrated a stronger correlation between BMD change and fracture risk reduction than the results based on the IPD analysis. In predicting the fracture risk reduction, the use of the average BMD gain (3%) observed in all studies in the calculations produced an overall fracture risk reduction very similar to the one observed clinically. In contrast, the use of a large BMD gain (8%) produced a substantially higher estimated fracture risk reduction and resulted in a high proportion of fracture risk reduction being attributed to BMD change.

Conclusions: Many factors may influence the outcome of meta-analyses, and caution should be used in interpreting the results of such analyses when exploring the relationship between BMD changes and fracture risk reduction with antiresorptive therapy of osteoporosis.
Statins Prevent Bisphosphonate-Induced γ,δ-T-Cell Proliferation and Activation In Vitro
Authors: Thompson Keith , Rogers Michael J
Published: April 2006

The acute phase response is the major adverse effect of intravenously administered N-BPs. In this study we show that N-BPs cause γ,δ-T-cell activation and proliferation in vitro by an indirect mechanism through inhibition of FPP synthase, an effect that can be overcome by inhibiting HMG-CoA reductase with a statin. These studies clarify the probable initial cause of the acute phase response to N-BP drugs and suggest a possible way of preventing this phenomenon.

Introduction: The acute phase response is the major adverse effect of intravenously administered nitrogen-containing bisphosphonate drugs (N-BPs), used in the treatment of metabolic bone diseases. This effect has recently been attributed to their action as non-peptide antigens and direct stimulation of γ,δ-T-cells. However, because N-BPs are potent inhibitors of farnesyl diphosphate (FPP) synthase, they could cause indirect activation of γ,δ-T-cells owing to the accumulation of intermediates upstream of FPP synthase in the mevalonate pathway, such as isopentenyl diphosphate/dimethylallyl diphosphate, which are known γ,δ-T-cell agonists.

Materials and Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers and treated with N-BP, statin, or intermediates/inhibitors of the mevalonate pathway for 7 days in the presence of interleukin (IL)-2. Flow cytometric analysis of the T-cell-gated population was used to quantify the proportion of γ,δ-T-cells in the CD3+ population.

Results and Conclusions: The ability of N-BPs to stimulate proliferation of CD3+ γ,δ-T-cells in human PBMC cultures matched the ability to inhibit FPP synthase. γ,δ-T-cell proliferation and activation (interferon γ [IFNγ] and TNFα release) was prevented by mevastatin or lovastatin, which inhibit HMG-CoA reductase upstream of FPP synthase and prevent the synthesis of isopentenyl diphosphate/dimethylallyl diphosphate. Desoxolovastatin, an analog of lovastatin incapable of inhibiting HMG-CoA reductase, did not overcome the stimulatory effect of N-BP. Furthermore, statins did not prevent the activation of γ,δ-T-cells by a synthetic γ,δ-T-cell agonist or by anti-CD3 antibody. Together, these observations show that N-BPs indirectly stimulate the proliferation and activation of γ,δ-T-cells caused by inhibition of FPP synthase and intracellular accumulation of isopentenyl diphosphate/dimethylallyl diphosphate in PBMCs. Because activation of γ,δ-T-cells could be the initiating event in the acute phase response to bisphosphonate therapy, co-administration of a statin could be an effective approach to prevent this adverse effect.
Actin-Related Protein 2/3 Complex Is Required for Actin Ring Formation
Authors: Hurst I Rita , Zuo Jian , Jiang Jin , Holliday L Shannon
Published: April 2006

Actin rings are vital for osteoclastic bone resorption, and actin-related protein 2/3 complex is a pivotal regulator of actin polymerization. Actin-related protein 2/3 complex was found in the podosomes of actin rings. A short interfering RNA knocked down expression of actin-related protein 2 in osteoclasts and disrupted actin rings, suggesting that the complex is crucial to actin ring formation.

Introduction: To resorb bone, osteoclasts form an extracellular acidic compartment segregated by a sealing zone. This is dependent on an actin ring that is composed of filamentous actin organized into dynamic structures called podosomes. The actin-related protein 2/3 (Arp2/3) complex is a vital regulator of actin polymerization. We tested whether the Arp2/3 complex is a component of actin rings and is important for actin ring formation.

Materials and Methods: Western blot analysis was used to determine levels of Arp2 and Arp3, two components of the Arp2/3 complex in osteoclast-like cells. Confocal microscopy studies using antibodies for immunocytochemistry demonstrated localization of Arp2/3 complex in osteoclasts. Short interfering RNA oligonucleotides (siRNAs) were made against Arp2 and used to knock down its expression.

Results: A 3-fold increase in Arp2 and Arp3 was detected during RANKL-induced differentiation of RAW 264.7 cells into osteoclast-like cells. Arp2/3 complex was concentrated in actin rings and enriched near the sealing zone. Arp2/3 complex co-localized with cortactin, a component of podosomes, but not vinculin, which surrounds podosomes. siRNA against Arp2, transfected into RAW 264.7 cells 5 days after stimulation with RANKL, reduced Arp2 protein levels 70% compared with cells transfected with ineffective siRNAs. Cytochemical characterization of RAW 264.7 osteoclast-like cells and marrow osteoclasts in which Arp2 was knocked down revealed fewer podosomes and no actin rings, although many cells remained well spread.

Conclusions: These data show that Arp2/3 complex is a component of actin rings and that the presence of Arp2/3 complex is vital to the formation of actin rings. In addition, the results show the use of siRNAs for the study of RAW 264.7 osteoclast-like cells.
Cooperation Between TGF-β and Wnt Pathways During Chondrocyte and Adipocyte Differentiation of Human Marrow Stromal Cells
Authors: Zhou Shuanhu , Eid Karim , Glowacki Julie
Published: April 2006

Human marrow stromal cells have the potential to differentiate to chondrocytes or adipocytes. We show interactions between TGF-β and Wnt signaling pathways during stimulation of chondrogenesis and inhibition of adipogenesis. Combining these signals may be useful in marrow stromal cell therapies.

Introduction: Human bone marrow stromal cells (hMSCs) have the potential to differentiate to lineages of mesenchymal tissues, including cartilage, fat, bone, tendon, and muscle. Agents like transforming growth factor (TGF)-β promote chondrocyte differentiation at the expense of adipocyte differentiation. In other processes, TGF-β and Wnt/wingless signaling pathways play major roles in controling certain developmental events and activation of specific target genes. We tested whether these pathways interact during differentiation of chondrocytes and adipocytes in human marrow stromal cells.

Materials and Methods: Both a line of human marrow stromal cells (KM101) and freshly isolated hMSCs were studied. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and macroarrays were used for analysis of the modulation of TGF-β1 on Wnt signaling-associated genes, chondrocyte differentiation genes, and TGFβ/bone morphogenetic protein (BMP) signaling-associated genes in KM101 cells. Early passage hMSCs obtained from 42- and 58-year-old women were used for the effects of TGF-β and/or Wnt (mimicked by LiCl) signals on chondrocyte and adipocyte differentiation in two-dimensional (2-D) cultures, 3-D pellet cultures, and collagen sponges.

Results: As indicated by macroarray, RT-PCR, and Western blot, TGF-β activated genes in the TGF-β/Smad pathway, upregulated Wnt2, Wnt4, Wnt5a, Wnt7a, Wnt10a, and Wnt co-receptor LRP5, and increased nuclear accumulation and stability of β-catenin in KM101 cells. TGF-β upregulated chondrocyte gene expression in KM101 cells and also stimulated chondrocyte differentiation and inhibited adipocyte differentiation in hMSCs, synergistically with Wnt signal. Finally, hMSCs cultured in 3-D collagen sponges were stimulated by TGF-β1 to express aggrecan and collagen type II mRNA, whereas expression of lipoprotein lipase was inhibited.

Conclusions: In summary, TGF-β stimulated chondrocyte differentiation and inhibited adipocyte differentiation of hMSCs in vitro. The activation of both TGF-β and Wnt signal pathways by TGF-β, and synergy between TGF-β and Wnt signals, supports the view that Wnt-mediated signaling is one of the mechanisms of TGF-β's effects on chondrocyte and adipocyte differentiation of hMSCs.
Skeletal Unloading Induces Resistance to Insulin-Like Growth Factor-I (IGF-I) by Inhibiting Activation of the IGF-I Signaling Pathways
Authors: Sakata Takeshi , Wang Yongmei , Halloran Bernard P , Elalieh Hashem Z , Cao Jay , Bikle Daniel D
Published: April 2006

We showed that unloading markedly diminished the effects of IGF-I to activate its signaling pathways, and the disintegrin echistatin showed a similar block in osteoprogenitor cells. Furthermore, unloading decreased αVβ3 integrin expression. These results show that skeletal unloading induces resistance to IGF-I by inhibiting activation of the IGF-I signaling pathways at least in part through downregulation of integrin signaling.

Introduction: We have previously reported that skeletal unloading induces resistance to insulin-like growth factor-I (IGF-I) with respect to bone formation. However, the underlying mechanism remains unclear. The aim of this study was to clarify how skeletal unloading induces resistance to the effects of IGF-I administration in vivo and in vitro with respect to bone formation.

Materials and Methods: We first determined the response of bone to IGF-I administration in vivo during skeletal unloading. We then evaluated the response of osteoprogenitor cells isolated from unloaded bones to IGF-I treatment in vitro with respect to activation of the IGF-I signaling pathways. Finally we examined the potential role of integrins in mediating the responsiveness of osteoprogenitor cells to IGF-I.

Results: IGF-I administration in vivo significantly increased proliferation of osteoblasts. Unloading markedly decreased proliferation and blocked the ability of IGF-I to increase proliferation. On a cellular level, IGF-I treatment in vitro stimulated the activation of its receptor, Ras, ERK1/2 (p44/42 MAPK), and Akt in cultured osteoprogenitor cells from normally loaded bones, but these effects were markedly diminished in cells from unloaded bones. These results were not caused by altered phosphatase activity or changes in receptor binding to IGF-I. Inhibition of the Ras/MAPK pathway was more impacted by unloading than that of Akt. The disintegrin echistatin (an antagonist of the αVβ3 integrin) blocked the ability of IGF-I to stimulate its receptor phosphorylation and osteoblast proliferation, similar to that seen in cells from unloaded bone. Furthermore, unloading significantly decreased the mRNA levels both of αV and β3 integrin subunits in osteoprogenitor cells.

Conclusion: These results indicate that skeletal unloading induces resistance to IGF-I by inhibiting the activation of IGF-I signaling pathways, at least in part, through downregulation of integrin signaling, resulting in decreased proliferation of osteoblasts and their precursors.
Prevention of Glucocorticoid-Induced Apoptosis in Osteocytes and Osteoblasts by Calbindin-D28k
Authors: Liu Yan , Porta Angela , Peng Xiaorong , Gengaro Kristen , Cunningham Earlene B , Li Hong , Dominguez Luis A , Bellido Teresita , Christakos Sylvia
Published: April 2006

This study show for the first time that calbindin-D28k can prevent glucocorticoid-induced bone cell death. The anti-apoptotic effect of calbindin-D28k involves inhibition of glucocorticoid induced caspase 3 activation as well as ERK activation.

Introduction: Recent studies have indicated that deleterious effects of glucocorticoids on bone involve increased apoptosis of osteocytes and osteoblasts. Because the calcium-binding protein calbindin-D28k has been reported to be anti-apoptotic in different cell types and in response to a variety of insults, we investigated whether calbindin-D28k could protect against glucocorticoid-induced cell death in bone cells.

Materials and Methods: Apoptosis was induced by addition of dexamethasone (dex; 10−6 M) for 6 h to MLO-Y4 osteocytic cells as well as to osteoblastic cells. Apoptosis percentage was determined by examining the nuclear morphology of transfected cells. Caspase 3 activity was evaluated in bone cells and in vitro. SELDI mass spectrometry (MS) was used to examine calbindin-D28k-caspase 3 interaction. Phosphorylation of calbindin-D28k was examined by32P incorporation as well as by MALDI-TOF MS. ERK activation was determined by Western blot.

Results: The pro-apoptotic effect of dex in MLO-Y4 cells was completely inhibited in cells transfected with calbindin-D28k cDNA (5.6% apoptosis in calbindin-D28k transfected cells compared with 16.2% apoptosis in vector-transfected cells, p < 0.05). Similar results were observed in osteoblastic cells. We found that dex-induced apoptosis in bone cells was accompanied by an increase in caspase 3 activity. This increase in caspase 3 activity was inhibited in the presence of calbindin-D28k. In vitro assays indicated a concentration-dependent inhibition of caspase 3 by calbindin-D28k (Ki = 0.22 μM). Calbindin-D28k was found to inhibit caspase 3 specifically because the activity of other caspases was unaffected by calbindin-D28k. The anti-apoptotic effect of calbindin-D28k in response to dex was also reproducibly associated with an increase in the phosphorylation of ERK 1 and 2, suggesting that calbindin-D28k affects more than one signal in the glucocorticoid-induced apoptotic pathway.

Conclusion: Calbindin-D28k, a natural non-oncogenic protein, could be an important target in the therapeutic intervention of glucocorticoid-induced osteoporosis.
BMD Is Reduced in HIV-Infected Men Irrespective of Treatment
Authors: Amiel C , Ostertag A , Slama L , Baudoin C , N'Guyen T , Lajeunie E , Neit-Ngeilh L , Rozenbaum W , De Vernejoul MC
Published: April 2006

Osteoporosis has be reported to be a complication of active antiretroviral therapy of HIV infection. We studied 148 HIV-infected men stratified according to their treatment. Our data show that these patients have an average 9% decreased BMD, irrespective of their treatment. Low body mass index and high resorption markers were associated with low bone density.

Introduction: Osteoporosis has been reported in HIV-infected (HIV+) patients, and it has been suggested that it may be linked to protease-inhibitor treatments (PI).

Materials and Methods: To assess this risk and to investigate its putative link with treatments, we compared the bone density of HIV+ men, who were either receiving treatment (including PI [PI+], n = 49; without PI [PI−], n = 51) or untreated (UT, n = 48). We included 81 age-matched control HIV-negative (HIV−) males (age, 40 ± 8 years).

Results: BMD adjusted for age (Z-score) was lower in the HIV+ patients at the lumbar spine (HIV+: −1.08 ± 1.21, HIV−: −0.06 ± 1.26, p < 0.001) and the femoral neck (HIV+: −0.39 ± 1.05, HIV−: 0.25 ± 0.87, p < 0.001). The prevalence of osteoporosis was 16% in HIV+ and 4% in HIV− subjects (p < 0.01). In the HIV+ subjects, the Z-score was correlated only to body mass index (r = 0.27 at lumbar spine and 0.35 at femoral neck). Untreated HIV+ patients had a negative Z-score (−0.82 ± 1.15 for the lumbar spine), which was not different from the one of treated HIV+ patients. In the PI+ and PI− groups, the Z-score did not depend on the presence of lipodystrophy or the proportion of fat in the abdomen and legs measured by DXA. Markers of bone remodeling were measured in the 132 HIV+ and 35 HIV− subjects. Compared with controls, HIV+ patients had lower bone alkaline phosphatase and higher urinary cross-laps/Cr, which was negatively correlated with the Z-score at both the femoral neck (r = −0.22) and lumbar spine (r = −0.21). TNFα was increased in untreated compared with treated HIV+ subjects and was not correlated to the Z-score.

Conclusion: Our cross-sectional study does not show any deleterious effect of the treatment but does indicate a decrease in bone density in HIV+ patients irrespective of the treatment. This low bone density is in part related to the low body weight and is associated with increased bone resorption.
Reproducibility and Side Differences of Mechanical Tests for Determining the Structural Strength of the Proximal Femur
Authors: Eckstein Felix , Wunderer Caecilia , Boehm Holger , Kuhn Volker , Priemel Mathias , Link Thomas M , Lochmüller Eva-Maria
Published: April 2006

In this experimental study, we evaluated the reproducibility error of mechanical strength tests of the proximal femur when simulating a fall on the trochanter. Based on side differences in femoral failure loads in 55 pairs of femora, we estimated the upper limit of the precision error to be 15% for the side impact test, whereas the intersubject variability was >40%.

Introduction: Mechanical tests are commonly used as the gold standard for determining one of the main functions of bones, that is, to provide mechanical strength. However, it is unknown what magnitude of error is associated with these tests. Here we investigate the precision error and side difference of a side impact test of the proximal femur.

Materials and Methods: BMC was measured using DXA in 54 pairs of femora from donors 79.0 ± 10.6 years of age. Bones were tested to failure, simulating a fall on the greater trochanter.

Results: Failure loads were 3951 ± 1659N (CV% = 42%) on the right and 3900 ± 1652N (CV% = 42%) on the left (no significant side difference). The average random difference of femoral BMC was 7 ± 7% and that of femoral failure loads was 17 ± 12%. The correlation between BMC and failure load was 79% (r2), but the association between side differences in failure load with those in BMC was only 4%. When confining the analysis to pairs with less than 5% differences in BMC (n = 31), side differences in failure loads were 15 ± 13%. When correcting failure loads for side differences of BMC, the difference was 16 ± 15%

Conclusions: These results suggest that the upper limit of the precision error for femoral strength tests is ∼15% in a side impact configuration. Given the large intersubject variability of failure loads, this test provides an efficient tool for determining the structural strength of the proximal femur in a fall.
Activation of Protease-Activated Receptor-2 Leads to Inhibition of Osteoclast Differentiation
Authors: Smith Rosealee , Ransjö Maria , Tatarczuch Liliana , Song Shu-Jun , Pagel Charles N , Morrison John R , Pike Robert N , Mackie Eleanor J
Published: April 2006

PAR-2 is expressed by osteoblasts and activated by proteases present during inflammation. PAR-2 activation inhibited osteoclast differentiation induced by hormones and cytokines in mouse bone marrow cultures and may protect bone from uncontrolled resorption.

Introduction: Protease-activated receptor-2 (PAR-2), which is expressed by osteoblasts, is activated specifically by a small number of proteases, including mast cell tryptase and factor Xa. PAR-2 is also activated by a peptide (RAP) that corresponds to the “tethered ligand” created by cleavage of the receptor's extracellular domain. The effect of activating PAR-2 on osteoclast differentiation was investigated.

Materials and Methods: Mouse bone marrow cultures have been used to investigate the effect of PAR-2 activation on osteoclast differentiation induced by parathyroid hormone (PTH), 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], and interleukin-11 (IL-11). Expression of PAR-2 by mouse bone marrow, mouse bone marrow stromal cell-enriched cultures, and the RAW264.7 osteoclastogenic cell line was demonstrated by RT-PCR.

Results: RAP was shown to inhibit osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11. Semiquantitative RT-PCR was used to investigate expression of mediators of osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11 in mouse bone marrow cultures and primary calvarial osteoblast cultures treated simultaneously with RAP. In bone marrow and osteoblast cultures treated with PTH, 1,25(OH)2D3, or IL-11, RAP inhibited expression of RANKL and significantly suppressed the ratio of RANKL:osteoprotegerin expression. Activation of PAR-2 led to reduced expression of prostaglandin G/H synthase-2 in bone marrow cultures treated with PTH, 1,25(OH)2D3, or IL-11. RAP inhibited PTH- or 1,25(OH)2D3-induced expression of IL-6 in bone marrow cultures. RAP had no effect on osteoclast differentiation in RANKL-treated RAW264.7 cells.

Conclusion: These observations indicate that PAR-2 activation inhibits osteoclast differentiation by acting on cells of the osteoblast lineage to modulate multiple mediators of the effects of PTH, 1,25(OH)2D3, and IL-11. Therefore, the role of PAR-2 in bone may be to protect it from uncontrolled resorption by limiting levels of osteoclast differentiation.
Bilateral Femoral Head Osteonecrosis After Septic Shock and Multiorgan Failure
Authors: Bolland Mark J , Hood Gill , Bastin Sonja T , King Alan R , Grey Andrew
Published: April 2006

A case of bilateral femoral head osteonecrosis after septic shock is presented. We suggest that the osteonecrosis was caused by ischemic insults to the proximal femora. The association between septic shock and osteonecrosis has not been previously reported.

Introduction: Osteonecrosis is an uncommon disorder characterized by the in situ death of bone. A diverse range of conditions has been associated with osteonecrosis. We present a case of bilateral femoral head osteonecrosis that occurred after an episode of septic shock.

Materials and Methods: A 66-year-old woman presented with a left-sided renal stone and a urinary tract infection. Her condition rapidly progressed to a life-threatening illness with septic shock complicated by multiorgan failure, which necessitated prolonged intensive care and inotropic support. She made a full recovery but 3 months later developed bilateral osteonecrosis of the femoral heads requiring bilateral total hip joint replacement.

Results and Conclusions: We propose that the osteonecrosis was caused by ischemic insults to the femoral heads as a result of the widespread systemic ischemia that occurred during her initial illness. To our knowledge, septic shock has not been previously described as a cause of osteonecrosis. Clinicians should be aware of this association, particularly in patients presenting with bone pain after episodes of sepsis.
Megakaryocyte-Osteoblast Interaction Revealed in Mice Deficient in Transcription Factors GATA-1 and NF-E2
Authors: Kacena Melissa A , Shivdasani Ramesh A , Wilson Kimberly , Xi Yougen , Troiano Nancy , Nazarian Ara , Gundberg Caren M , Bouxsein Mary L , Lorenzo Joseph A , Horowitz Mark C
Published: April 2006

Mice deficient in GATA-1 or NF-E2 have a 200–300% increase in bone volume and formation parameters. Osteoblasts and osteoclasts generated in vitro from mutant and control animals were similar in number and function. Osteoblast proliferation increased up to 6-fold when cultured with megakaryocytes. A megakaryocyte-osteoblast interaction plays a role in the increased bone formation in these mice.

Introduction: GATA-1 and NF-E2 are transcription factors required for the differentiation of megakaryocytes. Mice deficient in these factors have phenotypes characterized by markedly increased numbers of immature megakaryocytes, a concomitant drastic reduction of platelets, and a striking increased bone mass. The similar bone phenotype in both animal models led us to explore the interaction between osteoblasts and megakaryocytes.

Materials and Methods: Histomorphometry, μCT, and serum and urine biochemistries were used to assess the bone phenotype in these mice. Wildtype and mutant osteoblasts were examined for differences in proliferation, alkaline phosphatase activity, and osteocalcin secretion. In vitro osteoclast numbers and resorption were measured. Because mutant osteoblasts and osteoclasts were similar to control cells, and because of the similar bone phenotype, we explored the interaction between cells of the osteoblast lineage and megakaryocytes.

Results: A marked 2- to 3-fold increase in trabecular bone volume and bone formation indices were observed in these mice. A 20- to 150-fold increase in trabecular bone volume was measured for the entire femoral medullary canal. The increased bone mass phenotype in these animals was not caused by osteoclast defects, because osteoclast number and function were not compromised in vitro or in vivo. In contrast, in vivo osteoblast number and bone formation parameters were significantly elevated. When wildtype or mutant osteoblasts were cultured with megakaryocytes from GATA-1- or NF-E2-deficient mice, osteoblast proliferation increased over 3- to 6-fold by a mechanism that required cell-to-cell contact.

Conclusions: These observations show an interaction between megakaryocytes and osteoblasts, which results in osteoblast proliferation and increased bone mass, and may represent heretofore unrecognized anabolic pathways in bone.
Association of a Common Polymorphism in the Methylenetetrahydrofolate Reductase (MTHFR) Gene With Bone Phenotypes Depends on Plasma Folate Status
Authors: McLean Robert R , Karasik David , Selhub Jacob , Tucker Katherine L , Ordovas Jose M , Russo Giuseppina T , Cupples L Adrienne , Jacques Paul F , Kiel Douglas P
Published: April 2006

A study of a polymorphism in the MTHFR gene, plasma folate, and bone phenotypes in 1632 individuals revealed that the genotype effect on BMD and quantitative ultrasound was dependent on the level of folate. Our findings support the hypothesis that the association between an MTHFR polymorphism and bone phenotypes depends on folate status.

Introduction: Genome-wide screens using quantitative ultrasound (QUS) and BMD phenotypes have shown suggestive linkage on chromosome 1pter-1p36.3, a region containing the methylenetetrahydrofolate reductase (MTHFR) gene. Individuals homozygous (TT) for the MTHFR C677T polymorphism who have low plasma folate concentrations exhibit elevated plasma homocysteine (tHcy) concentrations that may compromise bone quality. We hypothesized that folate status might modify an association between the C677T polymorphism and bone, possibly by influencing homocysteine concentrations.

Materials and Methods: QUS (broadband ultrasound attenuation [BUA], speed of sound, and quantitative ultrasound index) of the heel and BMD of the hip and spine were measured in 1632 male and female members of the Framingham Offspring Study (1996–2001). Participants were assessed for plasma folate concentration and genotyped for the MTHFR C677T polymorphism. TT participants were compared with individuals in the CC + CT group using analysis of covariance.

Results: Adjusted mean QUS and BMD measures did not differ between C677T groups. Although all participants with plasma folate concentrations ≥4 ng/ml had ∼2% higher QUS and BMD than those with folate <4 ng/ml, the association disappeared after controlling for tHcy. Suggestive interactions between folate status and the C677T group (CC + CT versus TT) were found for hip BMD (p ≤ 0.05) and BUA (p = 0.11). Compared with CC + CT participants, TT individuals had lower mean BUA (p = 0.06) and Ward's area BMD (p = 0.08) within the folate <4 ng/ml group and significantly higher hip BMD (p ≤ 0.05) within the folate ≥4 ng/ml group. For both folate groups, TT participants had higher age-adjusted mean plasma tHcy versus CC + CT participants. Controlling for tHcy in these models did not affect the statistical significance of the interaction effects.

Conclusions: Our findings support the hypothesis that the association between the C677T MTHFR polymorphism and bone phenotypes depends on folate status. The mechanism mediating the association, however, remains unclear, but may be partially caused by homocysteine effects on bone.
Fracture Incidence in Polyostotic Fibrous Dysplasia and the McCune-Albright Syndrome
Authors: Leet Arabella I , Chebli Caroline , Kushner Harvey , Chen Clara C , Kelly Marilyn H , Brillante Beth A , Robey Pamela G , Bianco Paolo , Wientroub Shlomo , Collins Michael T
Published: April 2006

In patients with polyostotic fibrous dysplasia of bone, the peak incidence of fractures is during the first decade of life, followed by a decrease thereafter. Phosphaturia is associated with an earlier incidence and increased frequency of fractures.

Introduction: Fibrous dysplasia (FD) is a disorder involving either one (monostotic) or several bones (polyostotic FD [PFD] and sometimes is associated with cafe-au-lait hyperpigmentation of the skin and one or more hyperfunctioning endocrinopathies (McCune-Albright syndrome [MAS]). Both PFD and MAS are often associated with phosphaturia. Although fractures occur frequently in PFD/MAS, fracture incidence and the effect of age and co-existing metabolic abnormalities (endocrinopathy and/or phosphaturia) on fractures are ill defined.

Materials and Methods: We reviewed the medical records and examined the endocrine and phosphorus metabolism of 35 patients with PFD/MAS. We report on the age at which extremity fractures occurred and their location and treatment. The results of endocrine and phosphorus metabolism testing and associations between age of first fractures, number of fractures, fracture rate, and metabolic abnormalities were noted.

Results: The average follow-up was 14.2 years (range, 2–39 years), during which 172 fractures occurred. The number and sites of fractures were 103 femoral, 25 tibial, 33 humeral, and 11 forearm. Twenty-seven patients had PFD with one or more endocrinopathies and/or phosphaturia, and eight had PFD alone. The endocrinopathies included precocious puberty (n = 19), hyperthyroidism (n = 9), growth hormone excess (n = 6), and one patient each with Cushing syndrome and primary hyperparathyroidism. Twelve patients had phosphaturia. The peak rate of fractures occurred between 6 and 10 years of age and decreased thereafter. Patients with metabolic abnormalities sustained their first fracture at an earlier age (6.9 versus 16.6 years, p < 0.005) and had a higher lifetime rate of fractures (0.29 versus 0.08 fractures/year), relative to patients with PFD alone. Phosphaturia was the single metabolic dysfunction associated with both an earlier age of first fracture (5.1 versus 16.6 years, p < 0.05) and a greater lifetime fracture rate (0.35 versus 0.08 fractures/year, p < 0.05).

Conclusions: The occurrence of extremity fractures in FD peaks between 6 and 10 years of age and declines thereafter. Fractures occur earlier and more frequently in the presence of phosphaturia. These data have implications for long-term prognosis, clinical management, and interpretation of therapeutic interventions.
FGF-23 Is a Potent Regulator of Vitamin D Metabolism and Phosphate Homeostasis
Authors: Shimada Takashi , Hasegawa Hisashi , Yamazaki Yuji , Muto Takanori , Hino Rieko , Takeuchi Yasuhiro , Fujita Toshiro , Nakahara Kazuhiko , Fukumoto Seiji , Yamashita Takeyoshi
Published: April 2006

We analyzed the effects of an FGF-23 injection in vivo. FGF-23 caused a reduction in serum 1,25-dihydroxyvitamin D by altering the expressions of key enzymes for the vitamin D metabolism followed by hypophosphatemia. This study indicates that FGF-23 is a potent regulator of the vitamin D and phosphate metabolism.

Introduction: The pathophysiological contribution of FGF-23 in hypophosphatemic diseases was supported by animal studies in which the long-term administration of recombinant fibroblast growth factor-23 reproduced hypophosphatemic rickets with a low serum 1,25-dihydroxyvitamin D [1,25(OH)2D] level. However, there is no clear understanding of how FGF-23 causes these changes.

Materials and Methods: To elucidate the molecular mechanisms of the FGF-23 function, we investigated the short-term effects of a single administration of recombinant FGF-23 in normal and parathyroidectmized animals.

Results: An injection of recombinant FGF-23 caused a reduction in serum phosphate and 1,25(OH)2D levels. A decrease in serum phosphate was first observed 9 h after the injection and was accompanied with a reduction in renal mRNA and protein levels for the type IIa sodium-phosphate cotransporter (NaPi-2a). There was no increase in the parathyroid hormone (PTH) level throughout the experiment, and hypophosphatemia was reproduced by FGF-23 in parathyroidectomized rats. Before this hypophosphatemic effect, the serum 1,25(OH)2D level had already descended at 3 h and reached the nadir 9 h after the administration. FGF-23 reduced renal mRNA for 25-hydroxyvitamin D-1α-hydroxylase and increased that for 25-hydroxyvitamin D-24-hydroxylase starting at 1 h. In addition, an injection of calcitriol into normal mice increased the serum FGF-23 level within 4 h.

Conclusions: FGF-23 regulates NaPi-2a independently of PTH and the serum 1,25(OH)2D level by controlling renal expressions of key enzymes of the vitamin D metabolism. In conclusion, FGF-23 is a potent regulator of phosphate and vitamin D homeostasis.
Meta-Analysis of Molecular Association Studies: Vitamin D Receptor Gene Polymorphisms and BMD as a Case Study
Authors: Thakkinstian Ammarin , D'Este Catherine , Eisman John , Nguyen Tuan , Attia John
Published: April 2006

With the rise of molecular and genetic epidemiology, molecular association studies are increasingly common; however, meta-analysis of these studies has been a neglected area. This study performed a meta-analysis of the association of the vitamin D receptor (VDR) gene polymorphisms and BMD. We also highlight methodological issues that need to be resolved.

Introduction: With the rise of molecular and genetic epidemiology, molecular association studies are increasingly common; however, meta-analysis of these studies has been a neglected area. This study performed a meta-analysis of the association of vitamin D receptor (VDR) gene polymorphisms and BMD/osteoporosis and highlights methodological issues.

Materials and Methods: Studies published from 1994 to 2001 were identified through Medline using PubMed software. The reference lists of the articles retrieved were also reviewed. Where eligible papers had insufficient information, we contacted authors by mail (up to three mailings) for additional information. Any observational study, which tested the association between VDR BsmI genotypes and either BMD or osteoporosis at the femoral neck or spine in adult women, was included in the review. Data were extracted independently by two reviewers (AT and JA) using a standardized data extraction form.

Results: The B allele was significantly associated with BMD at the spine; it seemed to follow a recessive model, with the BB genotype having lower BMD than Bb/bb genotypes at baseline, which led to greater bone mineral loss over time. Highlighted methodological lessons included the need to check Hardy-Weinberg equilibrium and the importance of exploring heterogeneity, pooling data in a manner that is sensitive to genetic models, and avoiding multiple comparisons.

Conclusion: With the proliferation of molecular association studies, there will be an increased need to quantify the magnitude of the risk associated with genetic polymorphisms. This will likely entail meta-analytic methods, and this meta-analysis highlights some of the methodological issues that will need to be resolved.
Converse Relationship Between In Vitro Osteogenic Differentiation and In Vivo Bone Healing Elicited by Different Populations of Muscle-Derived Cells Genetically Engineered to Express BMP4
Authors: Peng Hairong , Usas Arvydas , Gearhart Brian , Olshanski Anne , Shen Hsain-Chung , Huard Johnny
Published: April 2006

In this study, we compared the use of primary muscle-derived osteoprogenitor cells (PP6 cells) for the delivery of BMP4 to improve bone healing to that of muscle-derived non-osteoprogenitor cells (PP1 cells). Surprisingly, the use of PP1 cells resulted in an improved outcome because of the lack of adverse responses to BMP4 involving cell differentiation, proliferation, and apoptosis.

Introduction: Although researchers frequently opt to use osteogenic cells for osteogenic bone morphogenetic protein (BMP)-based ex vivo gene therapy to improve bone healing, it remains unclear whether the osteogenic potential of a cellular vehicle affects the outcome of bone healing applications. Here we compared the use of muscle-derived non-osteoprogenitor cells (PP1 cells) to that of primary muscle-derived osteoprogenitor cells (PP6 cells) for the delivery of BMP4 to improve the healing of bone defects.

Materials and Methods: Two distinct populations of primary rat muscle-derived cells—PP1 and PP6—were selected, transduced with retroviral vectors to express BMP4 or a marker gene (LacZ), and implanted into critical-sized calvarial defects created in syngeneic rats. The bone healing was monitored radiographically and histologically at 7 and 14 weeks after implantation. Cellular responses to BMP4 were evaluated by alkaline phosphatase histochemical staining and RT-PCR of another osteogenic marker to indicate osteogenic differentiation, a cell proliferation assay and BrdU (bromodeoxyuridine) labeling to assess cell proliferation, and the TUNEL assay to determine apoptosis.

Results and Conclusions: In all animals (nine rats per group), transduced PP1 cells expressing BMP4 demonstrated significantly advanced healing compared with PP6 cells expressing BMP4 and control cells expressing LacZ. We found that constitutive BMP4 expression negatively impacted the in vitro proliferation and in vivo survival rates of PP6 cells, but not PP1 cells. BMP4 exposure also directly inhibited the proliferation and induced the apoptosis of PP6 cells, but not PP1 cells. The impairment in PP6 cell proliferation was directly associated with the osteogenic differentiation of these cells. These results indicate that PP1 cells are better suited than osteoprogenitor cells for use as cellular vehicles to deliver osteogenic BMP4 to improve bone healing and that cellular behavior in response to a particular gene can be used to predict the cells' performance as delivery vehicles in ex vivo gene therapy.