bluuebunny/crossref_metadata_2025 huggingface dataset:
Unmasking the Osteoinductive Effects of a G-Protein-Coupled Receptor (GPCR) Kinase (GRK) Inhibitor by Treatment With PTH(1–34)
Authors: Wang Liming , Quarles L Darryl , Spurney Robert F
Published: April 2006

The effects of GPCR systems in bone are regulated by a family of enzymes termed GRKs. We found that (1) GRK inhibition in osteoblasts has age-dependent effects on bone mass, and (2) the anabolic actions of GRK inhibition are revealed by treatment with PTH(1–34).

Introduction: The effects of G-protein-coupled receptor (GPCR) systems in bone are modulated by a family of enzymes termed GPCR kinases (GRKs). These enzymes directly phosphorylate GPCR substrate and desensitize receptor signaling. We previously found that expression of a GRK inhibitor in osteoblasts using transgenic (TG) technologies enhanced bone remodeling, and in turn, increased BMD in 6-week-old TG mice compared with non-TG littermate controls, presumably because of enhanced GPCR function. The aim of this study was to determine the age-dependent effects of the transgene.

Materials and Methods: BMD was monitored in TG mice and in controls at 6-week, 3-month, and 6-month time-points. To determine if the transgene enhanced responsiveness of bone to parathyroid hormone (PTH), we measured cyclic adenosine monophosphate (cAMP) generation by mouse calvaria ex vivo as well as the effects of treatment with PTH(1–34) on BMD, bone histomorphometry, and expression of the PTH-responsive gene RANKL in both TG mice and non-TG controls.

Results: Consistent with our previous findings, we found that BMD was increased in TG mice compared with controls at 6 weeks of age. The increase in BMD was most prominent in trabecular-rich lumbar spine and was not observed in cortical bone of the femoral shaft. In contrast to younger animals, however, BMD in older TG mice was not statistically different compared with non-TG mice at 3 months of age and was similar to non-TG animals at 6 months of age. The GRK inhibitor seemed to promote GPCR activation in older mice, however, because (1) PTH-induced cAMP generation by mouse calvaria ex vivo was enhanced in TG mice compared with controls, (2) GRK inhibition increased responsiveness of lumbar spine to the osteoinductive actions of PTH(1–34), and (3) the enhanced anabolic effect of PTH(1–34) was associated with increased expression of the PTH-responsive gene RANKL in calvaria of the TG animals. Bone histomorphometry confirmed that PTH(1–34) increased trabecular bone volume in TG mice and found that this increase in bone mass was caused by enhanced bone formation, predominantly as a result of an increase in the mineral apposition rate (MAR).

Conclusions: These data suggest that the anabolic effects of GRK inhibition are age dependent. The osteoinductive actions of the GRK inhibitor are, however, unmasked by treatment with PTH(1–34).
Safety and Efficacy of a Novel Salmon Calcitonin (sCT) Technology-Based Oral Formulation in Healthy Postmenopausal Women: Acute and 3-Month Effects on Biomarkers of Bone Turnover
Authors: Tankó László B , Bagger Yu Z , Alexandersen Peter , Devogelaer Jean-Pierre , Reginster Jean-Yves , Chick Rosalind , Olson Melvin , Benmammar Hakim , Mindeholm Linda , Azria Moise , Christiansen Claus
Published: April 2006

Oral administration of calcitonin could improve compliance to long-term treatment. Efficacy and safety of a novel oral formulation was assessed on 277 postmenopausal women. The results show (1) effective enteral absorption, (2) marked inhibition of bone resorption with minimal alteration of formation, and (3) reproducibility of responses over 3 months.

Introduction: We have recently introduced an Eligen technology-based oral formulation of salmon calcitonin (sCT) that effectively delivers the hormone to the circulation. The efficacy and safety during longer-term administration, however, has not been investigated in the target population.

Materials and Methods: This was a multicenter, randomized, double-blind, placebo-controlled, dose-ranging clinical trial including 277 healthy postmenopausal women 55-85 years of age. Women received treatment with either daily (0.15, 0.4, 1.0, or 2.5 mg) or intermittent doses (1.0 mg, every other day) of sCT combined with the delivery agent (8-[N-2-hydroxi-5-chloro-benzoyl]-amino-caprylic acid, 200 mg) or placebo for 3 months. All participants received 1000 mg calcium plus 400 IU vitamin D daily throughout the study. Efficacy parameters were the acute and/or pre-dose changes in serum and urinary C-terminal telopeptide of type I collagen (CTx), N-mid osteocalcin (OC), bone-specific alkaline phosphatase (BSALP), calcium, and parathyroid hormone (PTH) measured by established immunoassays.

Results: After the first dose, sCT evoked dose-dependent decreases in serum CTx (−60.8% to −81.8% from baseline) compared with placebo, reaching nadirs 2-3 h after drug intake, after which, gradual increases were observed. The simultaneous acute changes in OC were statistically nonsignificant. Area under the curve (AUC) of serum CTx responses at months 1 and 3 showed strong correlation with those at baseline (both r = 0.78, p < 0.001). At month 3, the placebo-corrected changes in the pre-dose value of serum and urinary CTx were significant only in the 1.0-mg dose group (−18.9% and −20.5%, respectively, p < 0.05). The placebo-corrected change in OC was −8.6 (p = 0.09), whereas the change in BSALP was −7.3 (p = 0.02). The oral formulation was well tolerated, with mild to moderate gastrointestinal and skin manifestations apparent mainly in the high-dose groups.

Conclusion: The results of this 3-month trial show that the novel Eligen technology-based oral formulation of sCT has potential to become a safe and effective treatment for postmenopausal bone loss. Future trials are needed to assess the impact of long-term administration on changes in BMD and fracture risk.
Disc Space Narrowing Is Associated With an Increased Vertebral Fracture Risk in Postmenopausal Women: The OFELY Study
Authors: Sornay-Rendu Elisabeth , Munoz Françoise , Duboeuf François , Delmas Pierre D
Published: April 2006

We have analyzed the relationship between spine osteoarthritis and fractures in the OFELY cohort. Despite a higher BMD associated with spine OA, the risk of fragility fractures is not reduced. Disc space narrowing is associated with an increased risk of vertebral fracture. These data indicate that the risk of osteoporotic fracture should not be underestimated in women with spine OA.

Introduction: Although osteoarthritis (OA) and osteoporosis both increase with age, their co-existence is uncommon. A higher BMD in OA is well documented, but a reduction of the fracture risk is still controversial. Our objective was to analyze the risk of fracture in postmenopausal women with spine OA.

Materials and Methods: In a cross-sectional study, spine OA was evaluated by lateral radiographs according to the method of Lane, and BMD was measured by DXA in 559 postmenopausal women from the OFELY cohort (mean age, 68 ± 8 years; range, 58–94 years) 8 years after their inclusion into the study. Previous fragility fractures, all confirmed by radiographs, were prospectively registered during the annual follow-up for 8 years, and vertebral fractures were evaluated with spine radiographs. Severity of OA was assessed by scoring on osteophytes and disc narrowing on a four-point scale from 0 (normal) to 3 (severe) and graded as 0 (normal), 1 (mild osteophyte and/or narrowing), or 2 (moderate or severe osteophyte and/or narrowing).

Results: Osteophytes and disc narrowing were present in 75% and 64%, respectively, of women at the lumbar spine and in 88% and 51%, respectively, at the thoracic spine, increasing with age. BMD of the spine, hip, and whole body increased with the severity of osteophytosis, whereas severity of narrowing was associated with a higher BMD only at the spine. Ninety-six fractures, including 48 vertebral fractures, occurred before OA assessment. No significant association was found between spine OA and all fragility fractures. In contrast, disc narrowing was associated with an increased risk of vertebral fracture with an odds ratio (95% CI) of 3.2 (1.1–9.3) after adjusting for age, body mass index, and BMD. The risk of vertebral fracture increased with the severity of disc narrowing. In comparison with the score 0, the odds ratio increased from 2.8 (0.9–8.7) to 4.6 (1.2–16.9) in women with mild to severe disc narrowing score.

Conclusions: Despite a higher BMD, women with spine OA do not have a reduced risk of fracture. Disc narrowing is associated with a significant increased vertebral fracture risk.
Increased VEGF Expression in the Epiphyseal Cartilage After Ischemic Necrosis of the Capital Femoral Epiphysis
Authors: Kim Harry KW , Bian Haikuo , Randall Timothy , Garces Amanda , Gerstenfeld Louis C , Einhorn Thomas A
Published: April 2006

Ischemic injury to the immature femoral head produces epiphyseal cartilage damage and cessation of endochondral ossification. This study suggests that VEGF facilitates the repair of the necrotic epiphyseal cartilage, which is essential for restoration of endochondral ossification and re-establishment of the growth of the immature femoral head after ischemic necrosis.

Introduction: Legg-Calve-Perthes disease (LCPD) is a childhood form of osteonecrosis that produces growth arrest of the secondary center of ossification. The cessation of growth is caused by ischemic damage to the hypertrophic zone of the epiphyseal cartilage where endochondral ossification normally occurs. The role of vascular endothelial growth factor (VEGF) in restoring endochondral ossification in the epiphyseal cartilage after ischemic necrosis was investigated in a piglet model of LCPD because the resumption of normal growth is important for maintaining the spherical shape of the femoral head.

Materials and Methods: Piglet femoral heads were assessed 24 h to 8 weeks after the surgical induction of ischemia. Western blot analysis, ribonuclease protection assay (RPA), immunohistochemistry, and in situ hybridization were performed.

Results: Western blot analysis and RPA showed increased VEGF protein and mRNA expression, respectively, in the epiphyseal cartilage of the infarcted heads compared with the contralateral normal heads. In the normal femoral heads, VEGF-immunoreactivity (VEGF-IR) and transcripts were observed in the hypertrophic zone of the epiphyseal cartilage. In the infarcted heads, VEGF-IR and transcripts were no longer observed in the hypertrophic zone because of diffuse cell death in that zone from ischemia. However, VEGF-IR and transcripts were observed in the proliferative zone above the necrotic hypertrophic zone. At 8 weeks, vascular granulation tissue invasion of the necrotic hypertrophic zone was observed with active resorption of the necrotic cartilage. In some areas where the necrotic cartilage was completely resorbed, restoration of endochondral ossification was observed. In these areas, VEGF transcripts were observed in the newly formed hypertrophic zone.

Conclusions: VEGF expression was increased, and its spatial expression was altered in the epiphyseal cartilage after ischemic necrosis of the immature femoral head. VEGF upregulation in the proliferative zone after ischemic damage may play a role in stimulating vascular invasion and granulation tissue formation in the necrotic hypertrophic zone of the epiphyseal cartilage. This may be an important step toward facilitating the resorption of the necrotic cartilage and restoration of endochondral ossification leading to further growth and development of the femoral head.
Osteoporosis and Fracture Risk in Women of Different Ethnic Groups
Authors: Barrett-Connor Elizabeth , Siris Ethel S , Wehren Lois E , Miller Paul D , Abbott Thomas A , Berger Marc L , Santora Arthur C , Sherwood Louis M
Published: April 2006

Osteoporosis and 1-year fracture risk were studied in 197,848 postmenopausal American women from five ethnic groups. Weight explained differences in BMD, except among blacks, who had the highest BMD. One SD decrease in BMD predicted a 50% increased fracture risk in each group. Despite similar relative risks, absolute fracture rates differed.

Introduction: Most information about osteoporosis comes from studies of white women. This study describes the frequency of osteoporosis and the association between BMD and fracture in women from five ethnic groups.

Materials and Methods: This study was made up of a cohort of 197,848 community-dwelling postmenopausal women (7784 blacks, 1912 Asians, 6973 Hispanics, and 1708 Native Americans) from the United States, without known osteoporosis or a recent BMD test. Heel, forearm, or finger BMD was measured, and risk factor information was obtained; 82% were followed for 1 year for new fractures. BMD and fracture rates were compared, adjusting for differences in covariates.

Results: By age 80, more than one-fifth of women in each ethnic group had peripheral BMD T scores <−2.5. Black women had the highest BMD; Asian women had the lowest. Only the BMD differences for blacks were not explained by differences in weight. After 1 year, 2414 new fractures of the spine, hip, forearm, wrist, or rib were reported. BMD at each site predicted fractures equally well within each ethnic group. After adjusting for BMD, weight, and other covariates, white and Hispanic women had the highest risk for fracture (relative risk ‘RR’ 1.0 ‘referent group’ and 0.95, 95% CI, 0.76, 1.20, respectively), followed by Native Americans (RR, 0.87; 95% CI, 0.57, 1.32), blacks (RR, 0.52; 95% CI, 0.38, 0.70), and Asian Americans (RR, 0.32; 95% CI, 0.15, 0.66). In age- and weight-adjusted models, each SD decrease in peripheral BMD predicted a 1.54 times increased risk of fracture in each ethnic group (95% CI, 1.48-1.61). Excluding wrist fractures, the most common fracture, did not materially change associations.

Conclusions: Ethnic differences in BMD are strongly influenced by body weight; fracture risk is strongly influenced by BMD in each group. Ethnic differences in absolute fracture risk remain, which may warrant ethnic-specific clinical recommendations.
Increased Bone Formation in Mice Lacking Apolipoprotein E
Authors: Schilling Arndt F , Schinke Thorsten , Münch Christian , Gebauer Matthias , Niemeier Andreas , Priemel Matthias , Streichert Thomas , Rueger Johannes M , Amling Michael
Published: April 2006

ApoE is a plasma protein that plays a major role in lipoprotein metabolism. Here we describe that ApoE expression is strongly induced on mineralization of primary osteoblast cultures. ApoE-deficient mice display an increased bone formation rate compared with wildtype controls, thereby showing that ApoE has a physiologic function in bone remodeling.

Introduction: Apolipoprotein E (ApoE) is a protein component of lipoproteins and facilitates their clearance from the circulation. This is confirmed by the phenotype of ApoE-deficient mice that have high plasma cholesterol levels and spontaneously develop atherosclerotic lesions. The bone phenotype of these mice has not been analyzed to date, although an association between certain ApoE alleles and BMD has been reported.

Materials and Methods: Primary osteoblasts were isolated from newborn mouse calvariae and mineralized ex vivo. A genome-wide expression analysis was performed during the course of differentiation using the Affymetrix gene chip system. Bones from ApoE-deficient mice and wildtype controls were analyzed using radiography, μCT imaging, and undecalcified histology. Cellular activities were assessed using dynamic histomorphometry and by measuring urinary collagen degradation products. Lipoprotein uptake assays were performed with125I-labeled triglyceride-rich lipoprotein-remnants (TRL-R) using primary osteoblasts from wildtype and ApoE-deficient mice. Serum concentrations of osteocalcin were determined by radioimmunoassay after hydroxyapatite chromatography.

Results: ApoE expression is strongly induced on mineralization of primary osteoblast cultures ex vivo. Mice lacking ApoE display a high bone mass phenotype that is caused by an increased bone formation rate, whereas bone resorption is not affected. This phenotype may be explained by a decreased uptake of triglyceride-rich lipoproteins by osteoblasts, resulting in elevated levels of undercarboxylated osteocalcin in the serum of ApoE-deficient mice.

Conclusion: The specific induction of ApoE gene expression during osteoblast differentiation along with the increased bone formation rate observed in ApoE-deficient mice shows that ApoE has a physiologic role as a regulator of osteoblast function.
Skeletal Localization and Neutralization of the SDF-1(CXCL12)/CXCR4 Axis Blocks Prostate Cancer Metastasis and Growth in Osseous Sites In Vivo
Authors: Sun Yan-Xi , Schneider Abraham , Jung Younghun , Wang Jianhua , Dai Jinlu , Wang Jingcheng , Cook Kevin , Osman Nadir I , Koh-Paige Amy J , Shim Hyusuk , Pienta Kenneth J , Keller Evan T , McCauley Laurie K , Taichman Russell S
Published: April 2006

To delineate the role of SDF-1 and CXCR4 in metastatic prostate cancer (CaP), positive correlations were established between SDF-1 levels and tumor metastasis. Neutralization of CXCR4 limited the number and the growth of intraosseous metastasis in vivo. Together, these in vivo metastasis data provide critical support that SDF-1/CXCR4 plays a role in skeletal metastasis.

Introduction: Previously we determined that the stromal-derived factor-1 (SDF-1)/CXCR4 chemokine axis is activated in prostate cancer (CaP) metastasis to bone. To delineate the role of SDF-1/CXCR4 in CaP, we evaluated SDF-1 levels in a variety of tissues and whether neutralization of SDF-1 prevented metastasis and/or intraosseous growth of CaPs.

Materials and Methods: SDF-1 levels were established in various mouse tissues by ELISA, immunohistochemistry, and in situ hybridization. To assess the role of SDF-1/CXCR4 in metastasis, bone metastases were established by administering CaP cells into the left cardiac ventricle of nude animals in the presence or absence of neutralizing CXCR4 antibody. The effect of SDF-1 on intraosseous growth of CaP cells was determined using intratibial injections and anti-CXCR4 antibodies and peptides.

Results: There was a positive correlation between the levels of SDF-1 and tissues in which metastatic CaP lesions were observed. SDF-1 levels were highest in the pelvis, tibia, femur, liver, and adrenal/kidneys compared with the lungs, tongue, and eye, suggesting a selective effect. SDF-1 staining was generally low or undetectable in the center of the marrow and in the diaphysis. SDF-1 mRNA was localized to the metaphysis of the long bones nearest to the growth plate where intense expression was observed near the endosteal surfaces covered by osteoblastic and lining cells. Antibody to CXCR4 significantly reduced the total metastatic load compared with IgG control-treated animals. Direct intratibial injection of tumor cells followed by neutralizing CXCR4 antibody or a specific peptide that blocks CXCR4 also decreased the size of the tumors compared with controls.

Conclusions: These data provide critical support for a role of SDF-1/CXCR4 in skeletal metastasis. Importantly, these data show that SDF-1/CXCR4 participate in localizing tumors to the bone marrow for prostate cancer.
Treatment of Painful Vertebral Fractures by Kyphoplasty in Patients With Primary Osteoporosis: A Prospective Nonrandomized Controlled Study
Authors: Kasperk Christian , Hillmeier Jochen , Nöldge Gerd , Grafe Ingo A , DaFonseca Katharina , Raupp Dorothea , Bardenheuer Hubert , Libicher Martin , Liegibel Ute Monika , Sommer Ulrike , Hilscher Ulrike , Pyerin Walter , Vetter Marcus , Meinzer Hans-Peter , Meeder Peter-Jürgen , Taylor Rod S , Nawroth Peter
Published: April 2006

This study investigates the effects of kyphoplasty on pain and mobility in patients with osteoporosis and painful vertebral fractures compared with conventional medical management.

Introduction: Pharmacological treatment of patients with primary osteoporosis does not prevent pain and impaired activity of patients with painful vertebral fractures. Therefore, we evaluated the clinical outcome after kyphoplasty in patients with vertebral fractures and associated chronic pain for >12 months.

Materials and Methods: Sixty patients with primary osteoporosis and painful vertebral fractures presenting for >12 months were included in this prospective, nonrandomized controlled study. Twenty-four hours before performing kyphoplasty, the patients self-determined their inclusion into the kyphoplasty or control group so that 40 patients were treated with kyphoplasty, whereas 20 served as controls. This study assessed changes in radiomorphology, pain visual analog scale (VAS) score, daily activities (European Vertebral Osteoporosis Study [EVOS] score), number of new vertebral fractures, and health care use. Outcomes were assessed before treatment and at 3 and 6 months of follow-up. All patients received standard medical treatment (1g calcium, 1000 IE vitamin D3, standard dose of oral aminobisphosphonate, pain medication, physical therapy).

Results: Kyphoplasty increased midline vertebral height of the treated vertebral bodies by 12.1%, whereas in the control group, vertebral height decreased by 8.2% (p = 0.001). Augmentation and internal stabilization by kyphoplasty resulted in a reduction of back pain. VAS pain scores improved in the kyphoplasty group from 26.2 ± 2 to 44.2 ± 3.3 (SD; p = 0.007) and in the control group from 33.6 ± 4.1 to 35.6 ± 4.1 (not significant), whereas the EVOS score increased in the kyphoplasty group from 43.8 ± 2.4 to 54.5 ± 2.7 (p = 0.031) and in the control group from 39.8 ± 4.5 to 43.8 ± 4.6 (not significant). The number of back pain-related doctor visits within the 6-month follow-up period decreased significantly after kyphoplasty compared with controls: mean of 3.3 visits/patient in the kyphoplasty group and a mean of 8.6 visits/patient in the control group (p = 0.0147).

Conclusions: The results of this study show significantly increased vertebral height, reduced pain, and improved mobility in patients after kyphoplasty. Kyphoplasty performed in appropriately selected osteoporotic patients with painful vertebral fractures is a promising addition to current medical treatment.
Deletion of Membrane-Bound Steel Factor Results in Osteopenia in Mice
Authors: Lotinun Sutada , Evans Glenda L , Turner Russell T , Oursler Merry Jo
Published: April 2006

To examine the functional role of membrane-bound SLF, we evaluated the growing skeletons of WT and SLF mutant (Sl/Sld) mice that do not produce this protein using DXA, bone histomorphometry, cell culture, and flow cytometry. Deletion of membrane-bound SLF delays bone growth, decreases bone mass and BMD, impairs osteoblast function, and increases osteoclast surface in young mice.

Introduction: Mutations at the murine steel locus lead to a defect in the development of hematopoietic stem cells, mast cells, and germ cells. Two isoforms of steel factor (SLF), soluble and membrane-associated, have been reported. Soluble SLF increases osteoclast formation and activity in cell culture. The effects of deletion of membrane-bound SLF on bone metabolism in mice have yet to be determined and are the subject of this study.

Materials and Methods: Five-, 7-, and 12-week-old male and 5-week-old female WCB6F1/J-KitlSl/KitlSl-d (Sl/Sld) mice and wildtype (WT) littermates were used. BMD and bone mass, growth, architecture, and turnover were evaluated by DXA (males and females) and histomorphometry (males only). Primary osteoblasts isolated from humeri of 5-week-old male WT and Sl/Sld mice were used to determine osteoblast function, and bone marrow cells from tibias and femurs of these mice were analyzed to determine cell surface expression of osteoclast precursors.

Results and Conclusions: Young Sl/Sld mice grew more slowly, had a reduced bone mass, and had shorter bones than WT littermates. Male mutants had significantly decreased whole body BMD in all age groups, largely because of a reduction in BMC. Tibial cross-sectional, cortical, and marrow area of cortical bone and cancellous bone volume was reduced in the mutants at all ages. The osteopenia in Sl/Sld was caused by increased osteoclast surface at all ages and decreased osteoblast surface at 5 weeks of age. [3H]thymidine incorporation studies showed that proliferation of osteoblasts derived from mutant mice was significantly suppressed (56%). Moreover, a decrease in mineralization was observed in Sl/Sld osteoblast culture. Fluorescence-activated cell sorting analysis of bone marrow cells from Sl/Sld mice revealed a 65% increase in the percentage of c-Fms+CD11b+RANK+ cells compared with WT controls. These findings suggest that membrane-bound SLF/c-Kit signaling plays a role in the regulation of peak bone mass.
Production of Prostaglandin D2 by Human Osteoblasts and Modulation of Osteoprotegerin, RANKL, and Cellular Migration by DP and CRTH2 Receptors
Authors: Gallant Maxime A , Samadfam Rana , Hackett Josette A , Antoniou John , Parent Jean-Luc , de Brum-Fernandes Artur J
Published: April 2006

Human osteoblasts produce PGD2, which acts on the DP receptor to decrease osteoprotegerin production and on the CRTH2 receptor to decrease RANKL expression and to induce osteoblast chemotaxis. These results indicate that activation of CRTH2 may lead to an anabolic response in bone.

Introduction: Whereas the actions of prostaglandin (PG)E2 as a modulator of bone and osteoblast function are relatively well characterized, little is known about PGD2 and bone metabolism. The objectives of this study were to determine if human osteoblasts can produce PGD2, which prostaglandin D2 synthases are implicated in this synthesis, to identify the PGD2 receptors (DP and CRTH2) on these cells and to characterize the biological effects resulting from their activation.

Materials and Methods: RT-PCR analysis and immunohistochemistry were used to detect PGD2 receptor and synthases in cultured human osteoblasts. Immunohistochemistry was used to identify the synthases and receptors in human bone tissue. Intracellular cAMP and calcium levels were determined to verify receptor activation. The cells were stimulated with PGD2 or the specific agonists BW 245C (DP) and DK-PGD2 (CRTH2), and the resulting effects on osteoprotegerin (OPG) secretion, RANKL expression, and chemotaxis were determined. Osteoblast production of PGD2 was evaluated by measuring PGD2 in the culture supernatants after stimulation with interleukin (IL)-1, TNF-α, PTH, vascular endothelial growth factor (VEGF), and insulin-like growth factor I (IGF-I).

Results: Human osteoblasts in culture generated PGD2 when stimulated. Both osteoblasts in culture and in situ present the lipocalin-type PGD2 synthase only. Both DP and CRTH2 receptors were present in human osteoblasts in culture and in situ. Stimulation of DP resulted in an increase in cAMP, whereas CRTH2 increased the intracellular calcium level. OPG production was reduced by 60% after DP receptor stimulation, whereas CRTH2 receptor stimulation decreased RANKL expression on human osteoblasts. As reported for other cell types, CRTH2 was a potent inducer of chemotaxis for human osteoblasts in culture.

Conclusions: Human osteoblasts in culture produce PGD2 under biologically relevant stimuli through the lipocalin-type PGD2 synthase (L-PGDS) pathway. As an autacoid, PGD2 can act on DP and CRTH2 receptors, both present on these cells. Specific activation of CRTH2 could lead directly and indirectly to an anabolic response in bone.
Non-Isomerized C-Telopeptide Fragments Are Highly Sensitive Markers for Monitoring Disease Activity and Treatment Efficacy in Paget's Disease of Bone
Authors: Alexandersen Peter , Peris Pilar , Guañabens Nuria , Byrjalsen Inger , Alvarez Luisa , Solberg Helene , Cloos Paul AC
Published: April 2006

A new resorption assay measuring non-isomerized collagen type I C-telopeptide fragments (α-α CTX) was evaluated in a cohort comprising 32 Pagetic patients and 48 healthy controls. α-α CTX was found to be a sensitive marker for assessing disease activity and monitoring treatment efficacy in Paget's disease of bone compared with isomerized CTX (β-β CTX) and a number of other established bone turnover markers.

Introduction: Collagen type I fragments are generated by resorbing osteoclasts, and some of them can be measured using a C-telopeptide (CTX) immunoassay. The C-telopeptide of collagen type I comprises a DG-motif susceptible to isomerization. In newly synthesized collagen, this motif is in the native form denoted α, but spontaneously converts to an isomerized form (β) during aging of bone. CTX fragments composed of at least two α CTX chains (α-α CTX) originating from degradation of newly formed bone can be determined in the urine using a newly developed sandwich ELISA. The aim of this study was to assess the ability of this marker to monitor disease activity and treatment efficacy in patients with Paget's disease compared with established bone turnover markers.

Materials and Methods: A total of 32 patients diagnosed with Paget's disease of bone was included in the study. All received 400 mg/day of oral tiludronate for 3 months. Urinary α-α CTX (U α-α CTX) was measured at baseline and at 1 and 6 months after discontinuation of therapy and in 48 untreated age-matched and healthy controls. Other markers of bone turnover, including urinary β-β CTX, N-terminal cross-linking telopeptide of type I collagen, and deoxypyridinoline, were also measured for comparison.

Results and Conclusions: The U α-α CTX marker showed a marked reduction (−82% and −77% at 1 and 6 months of treatment, respectively) in response to antiresorptive therapy in patients with Paget's disease. The response to treatment in this marker exceeded that of the other markers (p < 0.01). The α-α CTX marker also provided a high correlation (r = 0.89) to disease activity as assessed by scintigraphic activity index. In conclusion, α-α CTX seems to be a sensitive marker for assessing disease activity and monitoring treatment efficacy in Paget's disease.
Highly Ordered Interstitial Water Observed in Bone by Nuclear Magnetic Resonance
Authors: Wilson Erin E , Awonusi Ayorinde , Morris Michael D , Kohn David H , Tecklenburg Mary MJ , Beck Larry W
Published: April 2006

NMR was used to study the nanostructure of bone tissue. Distance measurements show that the first water layer at the surface of the mineral in cortical bone is structured. This water may serve to couple the mineral to the organic matrix and may play a role in deformation.

Introduction: The unique mechanical characteristics of bone tissue have not yet been satisfactorily connected to the exact molecular architecture of this complex composite material. Recently developed solid-state nuclear magnetic resonance (NMR) techniques are applied here to the mineral component to provide new structural distance constraints at the subnanometer scale.

Materials and Methods: NMR dipolar couplings between structural protons (OH− and H2O) and phosphorus (PO4) or carbon (CO3) were measured using the 2D Lee-Goldburg Cross-Polarization under Magic-Angle Spinning (2D LG-CPMAS) pulse sequence, which simultaneously suppresses the much stronger proton-proton dipolar interactions. The NMR dipolar couplings measured provide accurate distances between atoms, e.g., OH and PO4 in apatites. Excised and powdered femoral cortical bone was used for these experiments. Synthetic carbonate (∼2-4 wt%)-substituted hydroxyapatite was also studied for structural comparison.

Results: In synthetic apatite, the hydroxide ions are strongly hydrogen bonded to adjacent carbonate or phosphate ions, with hydrogen bond (O-H) distances of ∼1.96 Å observed. The bone tissue sample, in contrast, shows little evidence of ordered hydroxide. Instead, a very ordered (structural) layer of water molecules is identified, which hydrates the small bioapatite crystallites through very close arrangements. Water protons are ∼2.3-2.55 Å from surface phosphorus atoms.

Conclusions: In synthetic carbonated apatite, strong hydrogen bonds were observed between the hydroxide ions and structural phosphate and carbonate units in the apatite crystal lattice. These hydrogen bonding interactions may contribute to the long-range stability of this mineral structure. The biological apatite in cortical bone tissue shows evidence of hydrogen bonding with an ordered surface water layer at the faces of the mineral particles. This structural water layer has been inferred, but direct spectroscopic evidence of this interstitial water is given here. An ordered structural water layer sandwiched between the mineral and the organic collagen fibers may affect the biomechanical properties of this complex composite material.
Postmenopausal Bilateral Oophorectomy Is Not Associated With Increased Fracture Risk in Older Women
Authors: Antoniucci Diana M , Sellmeyer Deborah E , Cauley Jane A , Ensrud Kristine E , Schneider Jennifer L , Vesco Kimberly K , Cummings Steven R , Melton L Joseph
Published: April 2006

We studied whether oophorectomy performed after menopause is associated with an increased risk of hip or vertebral fractures in 6295 Study of Osteoporotic Fractures participants. There was no association between postmenopausal oophorectomy and the risk of hip or vertebral fractures.

Introduction: Bilateral oophorectomy after natural menopause has been associated with an increased risk of osteoporotic fractures, potentially because of a decline in serum estradiol and testosterone levels after the oophorectomy. We prospectively tested this hypothesis in the Study of Osteoporotic Fractures (SOF).

Materials and Methods: We studied 6295 white women ⩾65 years of age participating in the SOF who were not taking estrogen therapy at baseline. Hip fracture analyses included 708 hip fractures; vertebral fracture analyses included 267 incident vertebral fractures. Baseline serum estradiol and free testosterone values were available in a small subset of participants.

Results and Conclusion: There were no significant differences in age, weight, or BMD between the women who underwent postmenopausal oophorectomy (n = 583) and those who did not (n = 5712). Free testosterone levels were significantly lower among women who had a postmenopausal oophorectomy. A history of postmenopausal oophorectomy was not associated with an increased risk of hip (hazard ratio [HR] = 1.1; 95% CI = 0.9-1.5) or vertebral fracture (HR = 0.7; 95% CI = 0.5-1.2). The relationship between oophorectomy and hip fracture was not altered by adding serum estradiol level (HR = 1.3; 95% CI = 0.5-3.2) or serum free testosterone level (HR = 1.7; 95% CI = 0.8-3.7) to the model. In summary, postmenopausal oophorectomy was not associated with an increased risk of hip or vertebral fracture in this cohort. These results are in contrast to previous findings, suggesting that the relationship between postmenopausal oophorectomy and fractures is not fully elucidated and that incidental oophorectomy after menopause should still be considered carefully in each potential patient.
Adjusting Data to Body Size: A Comparison of Methods as Applied to Quantitative Trait Loci Analysis of Musculoskeletal Phenotypes
Authors: Lang Dean H , Sharkey Neil A , Lionikas Arimantas , Mack Holly A , Larsson Lars , Vogler George P , Vandenbergh David J , Blizard David A , Stout Joseph T , Stitt Joseph P , McClearn Gerald E
Published: April 2006

The aim of this study was to compare three methods of adjusting skeletal data for body size and examine their use in QTL analyses. It was found that dividing skeletal phenotypes by body mass index induced erroneous QTL results. The preferred method of body size adjustment was multiple regression.

Introduction: Many skeletal studies have reported strong correlations between phenotypes for muscle, bone, and body size, and these correlations add to the difficulty in identifying genetic influence on skeletal traits that are not mediated through overall body size. Quantitative trait loci (QTL) identified for skeletal phenotypes often map to the same chromosome regions as QTLs for body size. The actions of a QTL identified as influencing BMD could therefore be mediated through the generalized actions of growth on body size or muscle mass.

Materials and Methods: Three methods of adjusting skeletal phenotypes to body size were performed on morphologic, structural, and compositional measurements of the femur and tibia in 200-day-old C57BL/6J × DBA/2 (BXD) second generation (F2) mice (n = 400). A common method of removing the size effect has been through the use of ratios. This technique and two alternative techniques using simple and multiple regression were performed on muscle and skeletal data before QTL analyses, and the differences in QTL results were examined.

Results and Conclusions: The use of ratios to remove the size effect was shown to increase the size effect by inducing spurious correlations, thereby leading to inaccurate QTL results. Adjustments for body size using multiple regression eliminated these problems. Multiple regression should be used to remove the variance of co-factors related to skeletal phenotypes to allow for the study of genetic influence independent of correlated phenotypes. However, to better understand the genetic influence, adjusted and unadjusted skeletal QTL results should be compared. Additional insight can be gained by observing the difference in LOD score between the adjusted and nonadjusted phenotypes. Identifying QTLs that exert their effects on skeletal phenotypes through body size-related pathways as well as those having a more direct and independent influence on bone are equally important in deciphering the complex physiologic pathways responsible for the maintenance of bone health.
TRAF2 Is Essential for TNF-α-Induced Osteoclastogenesis
Authors: Kanazawa Kiyoshi , Kudo Akira
Published: April 2006

TRAF2-deficient mice show embryonic lethality, and we developed a new in vitro differentiation system to show the function of TRAF2 in osteoclastogenesis, in which osteoclast progenitors are derived from the fetal liver of TRAF2-deficient mice. Using this system, we showed that TRAF2 is required for TNF-α-induced osteoclastogenesis.

Introduction: TNF receptor-associated factor 2 (TRAF2) is a signal transducer for RANK and for two TNF receptor isotypes, TNFR1 and TNFR2. Because TRAF2-deficient mice show embryonic lethality, it has remained unclear whether TRAF2 is crucial in RANKL- or TNF-α-induced osteoclastogenesis.

Materials and Methods: Osteoclast progenitors derived from fetal liver were cultured in the presence of monocyte macrophage colony-stimulating factor (M-CSF), and flow cytometry for characterization of surface markers on these cells was performed. To examine the involvement of TRAF2 in osteoclast differentiation, we cultured osteoclast progenitors from TRAF2-deficient and wildtype mice with soluble RANKL or TNF-α in the presence of M-CSF, and counted the number of TRACP+ multinucleate cells formed. c-jun N-terminal kinase (JNK) and NF-κB activation in osteoclast progenitors was examined by Western blot analysis and electrophoretic mobility shift assay, respectively. Nuclear factor of activated T cells (NFATc1) expression and activation were analyzed by RT-PCR and immunofluorescence staining, respectively. To examine whether TRAF2 overexpression induced osteoclastogenesis, TRAF2 was overexpressed in osteoclast progenitors form wildtype bone marrow by retrovirus infection.

Results and Conclusions: Osteoclast progenitors from normal fetal liver, which were cultured with M-CSF, expressed surface molecules c-fms, Mac-1, and RANK, and could differentiate into TRACP+ multinucleate cells in the presence of soluble RANKL or TNF-α. RANKL-induced osteoclastogenesis gave a reduction of 20% in the progenitors from TRAF2-deficient mice compared with that of the cells from littermate wildtype mice, whereas TNF-α-induced osteoclastogenesis was severely impaired in the cells from the TRAF2-deficient mice. Only a few TRACP+ multinucleate cells were formed, and TNF-α-mediated activation of JNK, NF-κB, and NFATc1 was defective. TRAF2 overexpression induced differentiation of osteoclast progenitors from wildtype mice into TRACP+ multinucleate cells. These results suggest that TRAF2 plays an important role in TNF-α-induced osteoclastogenesis.
Relationship of Volumetric BMD and Structural Parameters at Different Skeletal Sites to Sex Steroid Levels in Men
Authors: Khosla Sundeep , Melton L Joseph , Robb Richard A , Camp Jon J , Atkinson Elizabeth J , Oberg Ann L , Rouleau Peggy A , Riggs B Lawrence
Published: April 2006

In a population-based, cross-sectional study, we related age-associated changes in vBMD and in bone structural parameters to circulating bioavailable estradiol and testosterone levels in men. Associations between these bone mass/structural parameters and sex steroid levels were progressively stronger with age. Our previously postulated “threshold” for skeletal estrogen deficiency was most evident at cortical sites.

Introduction: Serum sex steroids, particularly estrogen levels, are associated with bone mass in men, and previous work has suggested that there may be a “threshold” bioavailable estradiol (bio E2) level below which the male skeleton becomes estrogen deficient. However, previous studies addressing this issue have exclusively used DXA, which cannot separate trabecular from cortical bone or provide information on bone geometry or structure.

Materials and Methods: In an age-stratified population sample of 314 men (age, 22-91 years), we assessed volumetric BMD (vBMD) and bone geometry by QCT at the lumbar spine, femoral neck, distal radius, and distal tibia and related these to circulating bio E2 and bio testosterone (T) levels.

Results: Compared with young men (age, 20-39 years), middle-aged men (age, 40-59 years) had significantly lower bio T (−26%, p < 0.001) and bio E2 (−9%, p = 0.038) levels, and these decreases were even greater in the elderly men (age ≥ 60 years, −60% and −38% for bio T and bio E2, respectively, p < 0.001 for both). Reflecting their intact gonadal status, vBMD/structural parameters were not related to sex steroid levels in young men, whereas bio E2 levels were associated consistently with vBMD and variably with bone geometric parameters in the elderly men; middle-aged men showed associations with bio E2 and bio T at some sites. At all cortical sites, vBMD was associated with bio E2 at low (<30 pM, R = 0.27-0.41, p < 0.05-0.001) but not high (≥30 pM, R = −0.003 to 0.12, p = not significant) levels; no such differences were evident at trabecular sites.

Conclusions: In men, bio E2 is the most consistent predictor of vBMD and some bone geometric variables as assessed by QCT. We also extend our previous findings on a possible “threshold” for skeletal estrogen deficiency by showing that this is most evident for cortical sites.
Population Trends in BMD Testing, Treatment, and Hip and Wrist Fracture Rates: Are the Hip Fracture Projections Wrong?
Authors: Jaglal Susan B , Weller Iris , Mamdani Muhammad , Hawker Gillian , Kreder Hans , Jaakkimainen Liisa , Adachi Jonathan D
Published: April 2006

A worldwide epidemic of hip fractures has been predicted. Time trends in BMD testing, bone-sparing medications and hip and wrist fractures in the province of Ontario, Canada, were examined. From 1996 to 2001, BMD testing and use of bone-sparing medications increased each year, whereas despite the aging of the population, wrist and hip fracture rates decreased.

Introduction: If patients with osteoporosis are being diagnosed and effective treatments used with increasing frequency in the population, rates of hip and wrist fractures will remain stable or possibly decrease. We report here time trends in BMD testing, prescriptions for bone-sparing medications, hip and wrist fracture rates, and population projections of fracture rates to 2005 in the province of Ontario, Canada.

Materials and Methods: Ontario residents have universal access to Medicare. To examine time trends in BMD testing, all physician claims for DXA from 1992 to 2001 were selected from the Ontario Health Insurance Plan (OHIP) database. Trends in prescribing were examined from 1996 to 2003 using data from the Ontario Drug Benefit plan, which provides coverage to persons ≥65 years of age. Actual numbers of hip and wrist fractures were determined for 1992-2000 and population projections for 2001-2005 using time-series analysis. Wrist fractures were identified in the OHIP database and hip fractures through hospital discharge abstracts.

Results: From 1992 to 2001, the number of BMD tests increased 10-fold. There has been a steady increase in the number of persons filling prescriptions for antiresorptives (12,298 in 1996 to 225,580 in 2003) and the majority were for etidronate. For women, the rate of decline for wrist fractures is greater than that for hip fractures. The rate of hip fracture was fairly constant around 41 per 10,000 women ≥50 years between 1992 and 1996. In 1997, the hip fracture rate began to decrease, and the population projections suggest that this downward trend will continue to a rate of 33.1 per 10,000 in 2005.

Conclusions: Our findings suggest that fracture rates may be on the decline, despite the aging of the population, because of increased patterns of diagnosis and treatment for osteoporosis.
Inactivation of theNa-Cl Co-Transporter(NCC) Gene Is Associated With High BMD Through Both Renal and Bone Mechanisms: Analysis of Patients With Gitelman Syndrome andNccNull Mice
Authors: Nicolet-Barousse Laurence , Blanchard Anne , Roux Christian , Pietri Laurence , Bloch-Faure May , Kolta Sami , Chappard Christine , Geoffroy Valérie , Morieux Caroline , Jeunemaitre Xavier , Shull Gary E , Meneton Pierre , Paillard Michel , Houillier Pascal , De Vernejoul Marie-Christine
Published: April 2006

Chronic thiazide treatment is associated with high BMD. We report that patients and mice with null mutations in the thiazide-sensitive NaCl cotransporter (NCC) have higher renal tubular Ca reabsorption, higher BMD, and lower bone remodeling than controls, as well as abnormalities in Ca metabolism, mainly caused by Mg depletion.

Introduction: Chronic thiazide treatment decreases urinary Ca excretion (UVCa) and increases BMD. To understand the underlying mechanisms, Ca and bone metabolism were studied in two models of genetic inactivation of the thiazide-sensitive NaCl cotransporter (NCC): patients with Gitelman syndrome (GS) and Ncc knockout (Ncc−/−) mice.

Materials and Methods: Ca metabolism was analyzed in GS patients and Ncc−/− mice under conditions of low dietary Ca. BMD was measured by DXA in patients and mice, and bone histomorphometry was analyzed in mice.

Results: GS patients had low plasma Mg. They exhibited reduced UVCa, but similar serum Ca and GFR as control subjects, suggesting increased renal Ca reabsorption. Blood PTH was lower despite lower serum ionized Ca, and Mg repletion almost corrected both relative hypoparathyroidism and low UVCa. BMD was significantly increased in GS patients at both lumbar (+7%) and femoral (+16%) sites, and osteocalcin was reduced. In Ncc−/− mice, serum Ca and GFR were unchanged, but UVCa was reduced and PTH was elevated; Mg repletion largely corrected both abnormalities. Trabecular and cortical BMD were higher than in Ncc+/+ mice (+4% and +5%, respectively), and despite elevated PTH, were associated with higher cortical thickness and lower endosteal osteoclastic surface.

Conclusions: Higher BMD is observed in GS patients and Ncc−/− mice. Relative hypoparathyroidism (human) and bone resistance to PTH (mice), mainly caused by Mg depletion, can explain the low bone remodeling and normal/low serum Ca despite increased renal Ca reabsorption.