bluuebunny/crossref_metadata_2025 huggingface dataset:
Oropharyngeal Skeletal Disease Accompanying High Bone Mass and Novel LRP5 Mutation
Authors: Rickels Michael R , Zhang Xiafang , Mumm Steven , Whyte Michael P
Published: April 2006

Gain-of-function mutation in the gene encoding LRP5 causes high bone mass. A 59-year-old woman carrying a novel LRP5 missense mutation, Arg154Met, manifested skeletal disease affecting her oropharynx as well as dense bones, showing that exuberant LRP5 effects are not always benign.

Introduction: Gain-of-function mutation (Gly171Val) of LDL receptor-related protein 5 (LRP5) was discovered in 2002 in two American kindreds with high bone mass and benign phenotypes. In 2003, however, skeletal disease was reported for individuals from the Americas and Europe carrying any of six novel LRP5 missense mutations affecting the same LRP5 protein domain. Furthermore, in 2004, we described a patient with neurologic complications from dense bones and extensive oropharyngeal exostoses caused by the Gly171Val defect.

Materials and Methods: A 59-year-old woman was referred for dense bones. Three years before, mandibular buccal and lingual exostoses (osseous “tori”) were removed because of infections from food trapping between the teeth and exostoses. Maxillary buccal and palatal exostoses were asymptomatic. Radiographic skeletal survey showed marked thickening of the skull base and diaphyses of long bones (endosteal hyperostosis). BMD Z scores assessed by DXA were +8.5 and +8.7 in the total hip and L1-L4 spine (both ∼195% average control), respectively. LRP5 mutation analysis was carried out for the LRP5 domain known to cause high bone mass.

Results: Biochemical evaluation excluded most secondary causes of dense bones, and male-to-male transmission in her family indicated autosomal dominant inheritance. PCR amplification and sequencing of LRP5 exons 2-4 and adjacent splice sites revealed heterozygosity for a new LRP5 missense mutation, Arg154Met.

Conclusions: LRP5 Arg154Met is a novel defect that changes the same first “β-propeller” module as the eight previously reported LRP5 gain-of-function missense mutations. Arg154Met alters a region important for LRP5 antagonism by dickkopf (Dkk). Therefore, our patient's extensive oropharyngeal exostoses and endosteal hyperostosis likely reflect increased Wnt signaling and show that exuberant LRP5 effects are not always benign.
Influence of Glucocorticoids on Human Osteoclast Generation and Activity
Authors: Sivagurunathan Sutharshani , Muir Meloni M , Brennan Tara C , Seale J Paul , Mason Rebecca S
Published: April 2006

Using human peripheral blood mononuclear cells as osteoclast precursors, we showed that dexamethasone stimulated osteoclast generation at a pharmacological concentration but did not affect the life span of human osteoclasts. Dexamethasone also dose-dependently increased signals for osteoclastogenesis.

Introduction: Glucocorticoid-induced osteoporosis is a common and serious disease. Glucocorticoids predominantly affect osteoblast proliferation and life span. Much of the bone loss is caused by reduced bone formation, but there is also an element of increased bone resorption.

Materials and Methods: Human peripheral blood mononuclear cells were cultured on whale dentine and induced to differentiate to osteoclasts by RANKL and human macrophage-colony stimulating factor (M-CSF). Osteoclast activity was quantified by pit area. RANKL and osteoprotegerin (OPG) expression in osteoblasts were measured by real-time RT-PCR.

Results: In the early phase of osteoclast generation (0-16 days), cultures from two different donors showed that dexamethasone at 10−8 M increased pit area by 2.5-fold, whereas lower concentrations had no effect. At the highest dexamethasone concentration (10−7 M), pit area was reduced. In 21-day cultures from three other donors, a similar increase was seen with dexamethasone at 10−8 M. There was, however, no evidence of increased life span of osteoclasts with dexamethasone. In human primary osteoblasts, dexamethasone dose-dependently reduced OPG and increased RANKL expression as measured by quantitative real time RT-PCR.

Conclusion: These data provide some explanation at a cellular and molecular level for the observed increase in bone resorption seen in patients treated with glucocorticoids and indicate that there are clear direct effects of glucocorticoids on bone resorption in human cell systems that may differ from other species.
Retinoid Signaling Regulates CTGF Expression in Hypertrophic Chondrocytes With Differential Involvement of MAP Kinases
Authors: Shimo Tsuyoshi , Koyama Eiki , Sugito Hiroki , Wu Changshan , Shimo Satoko , Pacifici Maurizio
Published: April 2006

Retinoids are important for growth plate chondrocyte maturation, but their downstream effectors remain unclear. Recently, CTGF (CCN2) was found to regulate chondrocyte function, particularly in the hypertrophic zone. The goal of the study was to determine whether CTGF is a retinoid signaling effector molecule, how it is regulated, and how it acts.

Introduction: Using a combination of in vivo and in vitro approaches, we carried out a series of studies at the cellular, biochemical, and molecular level to determine whether and how retinoid signaling is related to expression and function of connective tissue growth factor (CTGF) in chondrocyte maturation and endochondral ossification.

Materials and Methods: Limbs of chick embryos in ovo were implanted with retinoic pan-antagonist RO 41-5253-filled beads, and phenotypic changes were assessed by in situ hybridization. CTGF gene expression and roles were tested in primary cultures of immature and hypertrophic chondrocytes. Cross-talk between retinoid signaling and other pathways was tested by determining endogenous levels of active ERK1/2 and p38 MAP kinases and phenotypic modulations exerted by specific antagonists of mitogen-activated protein (MAP) kinases and BMP signaling (Noggin).

Results: Interference with retinoid signaling blocked expression of CTGF and other posthypertrophic markers in long bone anlagen in vivo and hypertrophic chondrocyte cultures, whereas all-trans-retinoic acid (RA) boosted CTGF expression and even induced it in immature proliferating cultures. Exogenous recombinant CTGF stimulated chondrocyte maturation, but failed to do so in presence of retinoid antagonists. Immunoblots showed that hypertrophic chondrocytes contained sizable levels of phosphorylated ERK1/2 and p38 MAP kinases that were dose- and time-dependently increased by RA treatment. Experimental ERK1/2 inhibition led to a severe drop in baseline and RA-stimulated CTGF expression, whereas p38 inhibition increased it markedly. These responses were gene-specific, because the opposite was seen with other hypertrophic chondrocyte genes such as collagen X and RA receptor γ (RARγ). Tests with Noggin showed that RA induction of CTGF expression was negatively influenced by BMP signaling, whereas induction of collagen X expression was BMP-dependent.

Conclusions: Retinoids appear to have a preeminent role in controlling expression and function of CTGF in hypertrophic and posthypertrophic chondrocytes and do so with differential cooperation and intervention of MAP kinases and BMP signaling.
Ghrelin Directly Regulates Bone Formation
Authors: Fukushima Nobuhiro , Hanada Reiko , Teranishi Hitoshi , Fukue Yoshihiko , Tachibana Toshiaki , Ishikawa Hiroshi , Takeda Shu , Takeuchi Yasuhiro , Fukumoto Seiji , Kangawa Kenji , Nagata Kensei , Kojima Masayasu
Published: April 2006

To clarify the role of ghrelin in bone metabolism, we examined the effect of ghrelin in vitro and in vivo. Ghrelin and its receptor, GHS-R1a, were identified in osteoblasts, and ghrelin promoted both proliferation and differentiation. Furthermore, ghrelin increased BMD in rats. Our results show that ghrelin directly affects bone formation.

Introduction: Ghrelin is a gut peptide involved in growth hormone (GH) secretion and energy homeostasis. Recently, it has been reported that the adipocyte-derived hormone leptin, which also regulates energy homeostasis and opposes ghrelin's actions in energy homeostasis, plays a significant role in bone metabolism. This evidence implies that ghrelin may modulate bone metabolism; however, it has not been clarified. To study the role of ghrelin in skeletal integrity, we examined its effects on bone metabolism both in vitro and in vivo.

Materials and Methods: We measured the expression of ghrelin and growth hormone secretagogue receptor 1a (GHS-R1a) in rat osteoblasts using RT-PCR and immunohistochemistry (IHC). The effect of ghrelin on primary osteoblast-like cell proliferation was examined by recording changes in cell number and the level of DNA synthesis. Osteoblast differentiation markers (Runx2, collagen α1 type I [COLI], alkaline phosphatase [ALP], osteocalcin [OCN]) were analyzed using quantitative RT-PCR. We also examined calcium accumulation and ALP activity in osteoblast-like cells induced by ghrelin. Finally, to address the in vivo effects of ghrelin on bone metabolism, we examined the BMD of Sprague-Dawley (SD) rats and genetically GH-deficient, spontaneous dwarf rats (SDR).

Results: Ghrelin and GHS-R1a were identified in osteoblast-like cells. Ghrelin significantly increased osteoblast-like cell numbers and DNA synthesis in a dose-dependent manner. The proliferative effects of ghrelin were suppressed by [D-Lys3]-GHRP-6, an antagonist of GHS-R1a, in a dose-dependent manner. Furthermore, ghrelin increased the expression of osteoblast differentiation markers, ALP activity, and calcium accumulation in the matrix. Finally, ghrelin definitely increased BMD of both SD rats and SDRs.

Conclusions: These observations show that ghrelin directly stimulates bone formation.
Effect of Alendronate on the Age-Specific Incidence of Symptomatic Osteoporotic Fractures
Authors: Hochberg Marc C , Thompson Desmond E , Black Dennis M , Quandt Sara A , Cauley Jane , Geusens Piet , Ross Philip D , Baran Dan
Published: April 2006

Analyses of data from 3658 postmenopausal women with osteoporosis enrolled in the Fracture Intervention Trial showed that alendronate is effective in reducing the risk of symptomatic osteoporotic fractures across a spectrum of ages.

Introduction: Most osteoporosis studies examine the relative risk of fracture based on the entire duration of treatment. Because older patients tend to be at higher risk for osteoporosis-related fractures, this analysis examined the effect of alendronate treatment on the relative risk of fracture in terms of the age that patients attained during the study.

Materials and Methods: We studied 3658 postmenopausal women with osteoporosis 55-80 years of age at baseline enrolled in the Fracture Intervention Trial, a large randomized, double-blind, placebo-controlled study. Patients were treated with placebo or with alendronate at a daily dose of 5 mg for 2 years followed by 10 mg for an additional 1-2.5 years, and monitored for clinical fractures. Age, rather than study time, was the dynamic variable in our analysis.

Results: The relative risk reductions for hip, clinical spine, and wrist fractures were constant across age groups, without evidence of a decline at older ages. Specifically, alendronate reduced the risk of clinical fracture by 53% at the hip (relative risk [RR] = 0.47; 95% CI = 0.27-0.81; p < 0.01), 45% at the spine (RR = 0.55; 95% CI = 0.37-0.83; p < 0.01), and 31% at the wrist (RR = 0.69; 95% CI = 0.50-0.98; p = 0.038). In addition, alendronate produced a significant risk reduction of 40% (RR = 0.60; 95% CI = 0.47-0.77; p < 0.01) for the composite event of clinical hip, spine, and wrist fractures. As a consequence of the constant relative risk model, the absolute risk reduction with alendronate treatment increased with age because of the age-related increase in fracture risk in the placebo group. The absolute risk reduction for the composite event (hip, spine, and wrist fractures together) for alendronate treatment versus placebo was 65, 80, 111, and 161 women with fractures per 10,000 PYR for the 55 to <65, 65 to <70, 70 to <75, and 75-85 year age groups, respectively.

Conclusions: These data show that alendronate is effective in reducing the risk of symptomatic osteoporotic fractures across a spectrum of ages. The effectiveness is somewhat greater in patients with femoral neck T score ≤ −2.5 than in those with a T score ≤ −2.0.
Reduced Training Is Associated With Increased Loss of BMD
Authors: Valdimarsson Örnólfur , Alborg Henrik G , Düppe Henrik , Nyquist Fredrik , Karlsson Magnus
Published: April 2006

This 8-year controlled, follow-up study in 66 Swedish soccer women evaluated the effect of training and reduced training on BMD. The players who retired during the follow-up lost BMD in the femoral neck, whereas the controls did not.

Introduction: Physical activity during adolescence increases BMD, but whether the benefits are retained with reduced activity is controversial.

Materials and Methods: At baseline, DXA evaluated BMD in 48 active female soccer players with a mean age of 18.2 ± 4.4 (SD) years, in 18 former female soccer players with a mean age of 43.2 ± 6.2 years and retired for a mean of 9.4 ± 5.3 years, and in 64 age- and sex-matched controls. The soccer women were remeasured after a mean of 8.0 ± 0.3 years, when 35 of the players active at baseline had been retired for a mean of 5.3 ± 1.6 years.

Results and Conclusions: The players still active at follow-up had a higher BMD at baseline than the matched controls in the femoral neck (FN; 1.13 ± 0.19 versus 1.00 ± 0.13 g/cm2; p = 0.02). The yearly gain in BMD during follow-up was higher in the active players than in the controls in the leg (0.015 ± 0.006 versus 0.007 ± 0.012 g/cm2, p = 0.04). The soccer players who retired during follow-up had a higher BMD at baseline than the matched controls in the FN (1.13 ± 0.13 versus 1.04 ± 0.13 g/cm2; p = 0.005). The players that retired during follow-up lost BMD, whereas the controls gained BMD during the study period in the FN (−0.007 ± 0.01 versus 0.003 ± 0.02 g/cm2 yearly; p = 0.01). The soccer players already retired at baseline had higher BMD at study start than the matched controls in the leg (1.26 ± 0.09 versus 1.18 ± 0.10 g/cm2; p = 0.01). The former players who were retired at study start lost BMD, whereas the controls gained BMD during the study period in the trochanter (−0.006 ± 0.01 versus 0.004 ± 0.014 g/cm2 yearly; p = 0.01). This study shows that, in girls, intense exercise after puberty is associated with higher accrual of BMD, and decreased physical activity in both the short-term and long-term perspective is associated with higher BMD loss than in controls.
Growth Hormone Injections Improve Bone Quality in a Mouse Model of Osteogenesis Imperfecta
Authors: King Donna , Jarjoura David , McEwen Heather A , Askew Michael J
Published: April 2006

Systemic growth hormone injections increased spine and femur length in a mouse model of OI. Femur BMC, cross-sectional area, and BMD were increased. Smaller gains were produced in vertebral BMC and cross-sectional area. Biomechanical testing showed improvements to structural and material properties in the femur midshaft, supporting expanded testing of growth hormone therapy in children with OI.

Introduction: Osteoblasts in heterozygous Cola2oim mutant mice produce one-half the normal amounts of the α2 strand of type I procollagen. The mice experience a mild osteogenesis imperfecta (OI) phenotype, with femurs and vertebrae that require less force than normal to break in a biomechanical test.

Materials and Methods: Subcutaneous injections of recombinant human growth hormone (rhGH) or saline were given 6 days per week to oim/+ mice between 3 and 12 weeks of age, in a protocol designed to simulate a trial on OI children.

Results: rhGH injections promoted significant weight gain and skeletal growth compared with saline-treated control animals. Femur and spine lengths were increased significantly. Significant increases at the femur midshaft in cortical BMD (2.2%), BMC (15.5%), and cross-sectional area (13%) were produced by rhGH treatment. Increases in the same cortical bone parameters were measured in the metaphyseal region of the femur and in tail vertebrae, but lumbar vertebrae showed significant increases in BMC (9.6%) and cross-sectional area (10.1%) of trabecular bone. Three-point bending testing documented functional improvements to the femur midshafts. GH treatment produced significant increases in bone stiffness (23.7%), maximum load (30.8%), the energy absorbed by the femurs to the point of maximum load (44.5%), and the energy to actual fracture (40.4%). The ultimate stress endured by the bone material was increased by 14.1%.

Conclusions: Gains in bone length, cross-sectional area, BMD, BMC, structural biomechanical properties, and strength were achieved without directly addressing the genetic collagen defect in the mice. Results support expanded clinical testing of GH injections in children with OI.
Controlled Trial of Pamidronate in Children With Types III and IV Osteogenesis Imperfecta Confirms Vertebral Gains but Not Short-Term Functional Improvement
Authors: Letocha Anne D , Cintas Holly L , Troendle James F , Reynolds James C , Cann Christopher E , Chernoff Edith J , Hill Suvimol C , Gerber Lynn H , Marini Joan C
Published: April 2006

Bisphosphonates have been widely administered to children with OI based on observational trials. A randomized controlled trial of q3m intravenous pamidronate in children with types III and IV OI yielded positive vertebral changes in DXA and geometry after 1 year of treatment, but no further significant improvement during extended treatment. The treated group did not experience significantly decreased pain or long bone fractures or have increased motor function or muscle strength.

Introduction: Bisphosphonates, antiresorptive drugs for osteoporosis, are widely administered to children with osteogenesis imperfecta (OI). Uncontrolled pamidronate trials in OI reported increased BMD, vertebral coronal area, and mobility, and decreased pain. We conducted a randomized controlled trial of pamidronate in children with types III and IV OI.

Materials and Methods: This randomized trial included 18 children (4-13 years of age) with types III and IV OI. The first study year was controlled; 9 children received pamidronate (10 mg/m2/day IV for 3 days every 3 months). Four children in each group also received recombinant growth hormone (rGH) injections (0.06 mg/kg/day for 6 days/week). Seven children in the treatment group received pamidronate for an additional 6-21 months. All patients had L1-L4 DXA, spine QCT, spine radiographs, and musculoskeletal and functional testing.

Results: In the controlled phase, treated patients experienced a significant increase in L1-L4 DXA z score (p < 0.001) and increased L1-L4 midvertebral height (p = 0.014) and total vertebral area (p = 0.003) compared with controls. During extended treatment, DXA z scores and vertebral heights and areas did not increase significantly beyond the 12-month values. Fracture rate decreased significantly in the upper extremities (p = 0.04) but not the lower extremities (p = 0.09) during the first year of treatment. Gross motor function, muscle strength, and pain did not change significantly during the controlled or extended treatment phases.

Conclusions: A controlled trial confirmed the spine benefits of short-term pamidronate treatment in children with types III and IV OI. Pamidronate increased L1-L4 vertebral DXA and decreased vertebral compressions and upper extremity fractures. Vertebral measures did not improve during the extended treatment phase. The treatment group did not experience decreased lower extremity long bone fractures, significant improvement in growth, ambulation, muscle strength, or pain. There was substantial variability in individual response to treatment.
Human Bone Collagen Synthesis Is a Rapid, Nutritionally Modulated Process
Authors: Babraj John A , Smith Kenneth , Cuthbertson Daniel JR , Rickhuss Peter , Dorling James S , Rennie Michael J
Published: April 2006

We developed a direct assay of human bone collagen synthesis using [13C] or [15N] proline and applied it to determine the effects of feeding in young healthy men. Surprisingly, postabsorptive bone collagen synthesis is not sluggish, being ∼0.07%/h more rapid than that of muscle protein, and capable of being stimulated within 4 h of intravenous feeding by 66 ± 13%.

Introduction: All current methods for estimation of bone collagen turnover are indirect, depending on the assay of collagen “markers.” Our aim was to develop a direct method for human bone collagen synthesis to be used to study its physiology and pathology, and specifically, in the first instance, the effect of feeding.

Materials and Methods: We applied, over 2 h, flooding doses of [13C] and [15N] proline to label iliac crest bone collagen in eight young healthy men. The rate of collagen synthesis was determined as the rate of labeling of collagen hydroxyproline (assayed by gas chromatography-combustion-isotope ratio mass spectrometry in collagen extracted by differential solubility) compared with plasma proline labeling (assayed by gas chromatography-mass spectrometry). We also determined (in a second group of eight young healthy men) the effect of intravenous nutrition (glucose, lipid emulsion, and amino acids (in the ratio of 55%:30%:15% energy, respectively).

Results: Free bone proline labeling was 92 ± 6% of that of plasma proline, supporting the flooding dose assumption. Human iliac crest bone collagen is heterogeneous, with NaCl-EDTA, 0.5 M acetic acid, pepsin-acetic acid, and hot water-extractable pools being responsible for ∼1%, 3%, 8%, and 81% of content, respectively. The synthetic rates were 0.58 ± 0.1, 0.24 ± 0.05, 0.07 ± 0.02, and 0.06 ± 0.01%/h, respectively, giving an average rate of ∼0.066%/h. [13C] and [15N] proline gave identical results. Intravenous nutrition caused the disappearance of proline label from the procollagen pool and its increased appearance in the less extractable pools, suggesting nutritional stimulation of collagen processing.

Conclusion: The results show (1) that iliac crest bone collagen synthesis is faster than generally assumed and of the same order as muscle protein turnover and (2) that feeding increases synthesis by ∼66%. Given its ability to detect physiologically meaningful responses, the method should provide a new approach to studying the regulation of bone collagen turnover.
Comparison Insight Bone Measurements by Histomorphometry and μCT
Authors: Chappard Daniel , Retailleau-Gaborit Nadine , Legrand Erick , Baslé Michel Félix , Audran Maurice
Published: April 2006

Morphometric analysis of 70 bone biopsies was done in parallel by μCT and histomorphometry. μCT provided higher results for trabecular thickness and separation because of the 3D shape of these anatomical objects.

Introduction: Bone histomorphometry is used to explore the various metabolic bone diseases. The technique is done on microscopic 2D sections, and several methods have been proposed to extrapolate 2D measurements to the 3D dimension. X-ray μCT is a recently developed imaging tool to appreciate 3D architecture. Recently the use of 2D histomorphometric measurements have been shown to provide discordant results compared with 3D values obtained directly.

Material and Methods: Seventy human bone biopsies were removed from patients presenting with metabolic bone diseases. Complete bone biopsies were examined by μCT. Bone volume (BV/TV), Tb.Th, and Tb.Sp were measured on the 3D models. Tb.Th and Tb.Sp were measured by a method based on the sphere algorithm. In addition, six images were resliced and transferred to an image analyzer: bone volume and trabecular characteristics were measured after thresholding of the images. Bone cores were embedded undecalcified; histological sections were prepared and measured by routine histomorphometric methods providing another set of values for bone volume and trabecular characteristics. Comparison between the different methods was done by using regression analysis, Bland-Altman, Passing-Bablock, and Mountain plots.

Results: Correlations between all parameters were highly significant, but μCT overestimated bone volume. The osteoid volume had no influence in this series. Overestimation may have been caused by a double threshold used in μCT, giving trabecular boundaries less well defined than on histological sections. Correlations between Tb.Th and Tb.Sp values obtained by 3D or 2D measurements were lower, and 3D analysis always overestimated thickness by ∼50%. These increases could be attributed to the 3D shape of the object because the number of nodes and the size of the marrow cavities were correlated with 3D values.

Conclusion: In clinical practice, μCT seems to be an interesting method providing reliable morphometric results in less time than conventional histomorphometry. The correlation coefficient is not sufficient to study the agreement between techniques in histomorphometry. The architectural descriptors are influenced by the algorithms used in 3D.
Fibroblasts Express RANKL and Support Osteoclastogenesis in a COX-2-Dependent Manner After Stimulation With Titanium Particles
Authors: Wei Xiaochao , Zhang Xinping , Zuscik Michael J , Drissi M Hicham , Schwarz Edward M , O'Keefe Regis J
Published: April 2006

Synovial fibroblasts are possible mediators of osteolysis. Fibroblasts respond directly to titanium particles and increase RANKL expression through a COX-2/PGE2/EP4/PKA signaling pathway. Fibroblasts pretreated with titanium or PGE2 stimulated osteoclast formation, showing the functional importance of RANKL induction. Synovial fibroblasts and their activation pathways are potential targets to prevent osteolysis.

Introduction: Bone loss adjacent to the implant is a major cause of joint arthroplasty failure. Although the cellular and molecular response to microscopic wear debris particles is recognized as causative, little is known concerning role of synovial fibroblasts in these events.

Materials and Methods: Murine embryonic fibroblasts and knee synovial fibroblasts in culture stimulated with titanium particles were examined by FACS, real time RT-PCR, Northern blot, and Western blot for expressions of vascular cell adhesion molecule (VCAM)1, RANKL, cyclooxygenase (COX)-1, and COX-2, and the four prostaglandin E2 (PGE2) receptor isoforms. Experiments were performed in the presence and absence of COX inhibitors, protein kinase A (PKA) and protein kinase C (PKC) inhibitors, and various EP receptor agonists. Osteoclast formation was examined in co-cultures of pretreated glutaraldehyde-fixed fibroblasts and primary murine spleen cells treated with macrophage-colony stimulating factor (M-CSF) for 7-days.

Results: TNF-α stimulated VCAM1 expression, consistent with a synovial fibroblast phenotype. Titanium particles stimulated RANKL gene and protein expressions in fibroblasts in a dose-dependent manner. Gene expression was increased 5-fold by 4 h, and protein levels reached a maximum after 48 h. Within 1 h, titanium particles also induced COX-2 mRNA and protein levels, whereas both indomethacin and celecoxib blocked the stimulation of RANKL, suggesting a COX-2-mediated event. Furthermore, PGE2 induced RANKL gene and protein expression and rescued RANKL expression in titanium-treated cultures containing COX-2 inhibitors. Fibroblast cultures pretreated with either PGE2 or titanium particles enhanced osteoclast formation, indicating the functional importance of RANKL induction. EP4 was the most abundant PGE2 receptor isoform, EP1 and EP2 were expressed at low levels, and EP3 was absent. The EP1 selective agonist iloprost and the EP2 selective agonist butaprost minimally stimulated RANKL. In contrast, the EP2 and EP4 agonist misoprostol induced RANKL to a magnitude similar to PGE2. Finally, PKA antagonism strongly repressed RANKL stimulation by PGE2.

Conclusion: Fibroblasts respond directly to titanium particles and increase RANKL expression through a COX-2/PGE2/EP4/PKA signaling pathway. Thus, the synovial fibroblast is important mediator of osteolysis and target for therapeutic strategies.
Smoking and the Risk of Fracture in Older Men
Authors: Olofsson Helena , Byberg Liisa , Mohsen Rawya , Melhus Hakan , Lithell Hans , Michaëlsson Karl
Published: April 2006

The role of smoking on fracture risk in older men was studied within a longitudinal population-based cohort study. Using time-dependent exposure information and analysis, smoking was detected to be a stronger, dose-dependent and a more long lasting risk factor for fracture than has previously been estimated.

Introduction: Although several studies have indicated a negative influence of smoking on fracture risk in women, there are few studies in men. No study in either sex has considered that smoking exposure may vary during follow-up in a cohort study. There is a need for a prospective study with repeated measures to analyze smoking exposure and fracture risk in men.

Materials and Methods: A total of 2322 men, 49-51 years of age, were enrolled in a longitudinal, population-based cohort study. Smoking status and other lifestyle habits were established at baseline and additionally at 60, 70, and 77 years of age. One or more fractures were documented in 272 men during 30 years of follow-up. Cox proportional hazards regression was used to determine the rate ratio (RR) of fracture according to time-dependent smoking habits and covariates.

Results: The overall adjusted fracture risk was increased in current (RR, 2.71; 95% CI, 1.86-3.95) and former smokers (RR, 1.66, 95% CI; 1.18-2.34), and persistent until 30 years after cessation. Among current smokers, the adjusted risk of any fracture increased by 30% (95% CI, 6-58%) for every 5 g of tobacco smoked each day. Smoking duration did not substantially influence fracture risk in either current or former smokers. One-half (52%; 95% CI, 35-65%) of all fractures were attributable to current smoking.

Conclusions: Tobacco smoking seems to be a long-lasting major risk factor for fracture in older men, and the risks depends both on recency of smoking and on the daily amount of tobacco smoked, rather than smoking duration.
Interleukin-11 Receptor Signaling Is Required for Normal Bone Remodeling
Authors: Sims Natalie A , Jenkins Brendan J , Nakamura Akira , Quinn Julian MW , Li Ruili , Gillespie Matthew T , Ernst Matthias , Robb Lorraine , Martin T John
Published: April 2006

IL-6 and -11 regulate bone turnover and have been implicated in estrogen deficiency-related bone loss. In this study, deletion of IL-11 signaling, but not that of IL-6, suppressed osteoclast differentiation, resulting in high trabecular bone volume and reduced bone formation. Furthermore, IL-11 signaling was not required for the effects of estradiol or estrogen deficiency on the mouse skeleton.

Introduction: Interleukin (IL)-6 and -11 stimulate osteoclastogenesis and bone formation in vitro and have been implicated in bone loss in estrogen deficiency. Because of their common use of the gp130 co-receptor signaling subunit, the roles of these two cytokines are linked, and each may compensate for the absence of the other to maintain trabecular bone volume and bone cell differentiation.

Materials and Methods: To determine the interactions in bone between IL-11 and IL-6 in vivo and whether IL-11 is required for normal bone turnover, we examined the bone phenotype of mature male and female IL-11 receptor knockout mice (IL-11Rα1−/−) and compared with the bone phenotype of IL-6−/− mice and mice lacking both IL-6 and IL-11Rα. To determine whether IL-11 is required for the effects of estrogen on trabecular bone, mature IL-11Rα1−/− mice were ovariectomized and treated with estradiol.

Results: In both male and female IL-11Rα1−/− mice, trabecular bone volume was significantly higher than that of wildtype controls. This was associated with low bone resorption and low bone formation, and the low osteoclast number generated by IL-11Rα1−/− precursors was reproduced in ex vivo cultures, whereas elevated osteoblast generation was not. Neither trabecular bone volume nor bone turnover was altered in IL-6−/− mice, and compound IL-6−/−:IL-11Rα1−/− mice showed an identical bone phenotype to IL-11Rα1−/− mice. The responses of IL-11Rα1−/− mice to ovariectomy and estradiol treatment were the same as those observed in wildtype mice.

Conclusions: IL-11 signaling is clearly required for normal bone turnover and normal trabecular bone mass, yet not for the effects of estradiol or estrogen deficiency on the skeleton. In the absence of IL-11Rα, increased trabecular bone mass seems to result from a cell lineage-autonomous reduction in osteoclast differentiation, suggesting a direct effect of IL-11 on osteoclast precursors. The effects of IL-11Rα deletion on the skeleton are not mediated or compensated for by changes in IL-6 signaling.
Effects of Intravenous Pamidronate Treatment in Infants With Osteogenesis Imperfecta: Clinical and Histomorphometric Outcome
Authors: Munns Craig FJ , Rauch Frank , Travers Rose , Glorieux Francis H
Published: April 2006

Clinical and histomorphometric outcome was compared between children with OI who had received pamidronate since infancy and age-matched patients who had never received pamidronate. Pamidronate was associated with improved vertebral shape and mass, higher cortical width, increased cancellous bone volume, and suppressed bone turnover.

Introduction: Observations in small patient series indicate that infants with severe osteogenesis imperfecta (OI) benefit from treatment with cyclical intravenous pamidronate. However, detailed analyses of outcome are lacking for this age group.

Materials and Methods: Clinical outcome was evaluated in 29 children with OI types I (n = 3), III (n = 14), or IV (n = 12) who started pamidronate therapy before 2 years of age (age at treatment onset: median, 6 months; range, 2 weeks to 23 months) and who had completed 3 years of treatment (total annual pamidronate dose, 9 mg/kg). They were compared with a historical control group of 29 untreated children with severe OI who were matched for OI type and age at the 3-year treatment time-point. In addition, iliac bone histomorphometry was compared between 24 pamidronate-treated patients and 24 age-matched OI patients who had not received pamidronate.

Results: Morphometric evaluation of lumbar vertebrae (L1-L4) showed that the shape of vertebral bodies was better preserved in pamidronate-treated patients. This was accompanied by significantly higher lumbar spine areal and volumetric BMD (+110 and +96%, respectively) and a larger vertebral bone volume (+26%) on densitometry. Regarding mobility function, the Pediatric Evaluation of Disability Inventory gross motor score was 50% greater in the pamidronate group (p < 0.001). Iliac bone histomorphometry showed 61% higher cortical width and 89% higher cancellous bone volume in pamidronate-treated patients. Bone formation rate per bone surface in the pamidronate group was only 17% that of untreated patients.

Conclusions: In conclusion, this study suggests that cyclical pamidronate treatment started in infancy leads to improved bone strength and better gross motor function but also suppresses bone turnover markedly. It is therefore prudent to reserve pamidronate treatment to infant OI patients who present with a moderate to severe phenotype.
Examining Bone Surfaces Across Puberty: A 20-Month pQCT Trial
Authors: Kontulainen Saija A , Macdonald Heather M , Khan Karim M , McKay Heather A
Published: April 2006

This follow-up study assessed sex differences in cortical bone growth at the tibial midshaft across puberty. In both sexes, periosteal apposition dominated over endosteal resorption. Boys had a greater magnitude of change at both surfaces, and thus, a greater increase in bone size across puberty. Relative increase in cortical bone area was similar between sexes.

Introduction: Generally, sex differences in bone size become most evident as puberty progresses. This was thought to be caused, in part, by greater periosteal apposition in boys, whereas endosteal apposition prevailed in girls. However, this premise is based on evidence from cross-sectional studies and planar measurement techniques. Thus, our aim was to prospectively evaluate sex-specific changes in cortical bone area across puberty.

Materials and Methods: We used pQCT to assess the tibial midshaft (50% site) at baseline and final (20 months) in girls (N = 68) and boys (N = 60) across early-, peri-, and postpuberty. We report total bone cross-sectional area (ToA, mm2), cortical area (CoA, mm2), marrow cavity area (CavA, mm2), and CoA/ToA ratio.

Results: Children were a mean age of 11.9 ± 0.6 (SD) years at baseline. At the tibia, CoA ranged from 230 ± 44, 261 ± 50, and 258 ± 46 in early-, peri-, and postpubertal girls. In boys, comparable values were 223 ± 36 (early), 264 ± 38 (peri), and 281 ± 77 (postpubertal). There was no sex difference for ToA or CoA at baseline. Increase in ToA and CoA was, on average, 10% greater for boys than girls across maturity groups. The area of the marrow cavity increased in all groups, but with considerable variability. The increase in CavA was significantly less for girls than boys in the early- and postpubertal groups. Change in CoA/ToA was similar between sexes across puberty.

Conclusion: Both sexes showed a similar pattern of change in CoA at the tibial midshaft, where periosteal apposition dominated over endosteal resorption. Boys showed a greater magnitude of change at both surfaces, and thus, showed a greater increase in bone size across puberty. The relative increase in cortical area was similar between sexes. These pQCT findings provide no evidence for endosteal apposition in postmenarchal girls.
α-Lipoic Acid Inhibits TNF-α-Induced Apoptosis in Human Bone Marrow Stromal Cells
Authors: Byun Chang-Hyun , Koh Jung-Min , Kim Dong Kwan , Park Seung-Il , Lee Ki-Up , Kim Ghi Su
Published: April 2006

TNF-α is an important mediator of bone loss. In the HS-5 hBMSC, TNF-α and H2O2 increased intracellular ROS levels and induced cell apoptosis through activation of caspases, JNK and NF-κB. α-Lipoic acid prevented these changes induced by TNF-α and H2O2, suggesting its potential therapeutic applications in attenuating bone loss.

Introduction: Oxidative stress is an important mediator of bone loss. TNF-α, which plays a critical role in the bone loss after menopause, has been shown to increase intracellular oxidative stress. Because oxidative stress is associated with cell death, we analyzed the apoptotic effects of TNF-α and H2O2 on human bone marrow stromal cells (hBMSCs). We also examined the protective effects of an important biological thiol antioxidant, α-lipoic acid (α-LA), against TNF-α- and H2O2-induced apoptosis.

Materials and Methods: Using the HS-5 hBMSC cell line, we tested whether TNF-α-induced apoptosis was mediated by the generation of excessive reactive oxygen species (ROS). Apoptosis was determined by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay, trypan blue exclusion assay, quantitation of histone-associated DNA fragments in cytosol, and the activation of caspases. The mechanisms mediating these apoptotic effects were determined by Western blotting and enzyme immunoassay.

Results: Both TNF-α and H2O2 increased intracellular ROS levels, reduced total cellular glutathione levels, activated caspases-3, -9, and -8, and enhanced hBMSC apoptosis. The activation of c-jun N-terminal kinase (JNK) and NF-κB mediated these apoptotic effects. Pretreatment of cells with α-LA prevented these changes induced by TNF-α and H2O2.

Conclusions: Our data show that TNF-α increases intracellular ROS in hBMSC and that TNF-α and H2O2 induce apoptosis in hBMSC through the activation of JNK and NF-κB. Our findings also suggest that α-LA may have therapeutic applications in halting or attenuating bone loss associated with increased oxidative stress.
An Autosomal Dominant High Bone Mass Phenotype in Association With Craniosynostosis in an Extended Family Is Caused by an LRP5 Missense Mutation
Authors: Kwee Mei Lan , Balemans Wendy , Cleiren Erna , Gille Johan JP , Van Der Blij Frits , Sepers Jan M , Van Hul Wim
Published: April 2006

Gain-of-function mutations in LRP5 have been shown to cause high BMD disorders showing variable expression of some clinical symptoms, including torus palatinus and neurological complications. In an extended family, we were able to add craniosynostosis and developmental delay to the clinical spectrum associated with LRP5 mutations.

We report on an extended four-generation family with 13 affected individuals (7 men and 6 women) in which an autosomal dominant type of osteosclerosis segregates. Osteosclerosis was most pronounced in the cranial base and calvarium, starting in early childhood with variable expression and a progressive character. Craniosynostosis at an early age was reported in four affected family members (two males and two females). The patients also presented with dysmorphic features (macrocephaly, brachycephaly, wide and high forehead, hypertelorism, prominent cheekbones, prominent jaw). They have normal height and proportions. Neurological complications like entrapment of cranial nerves resulting in optical nerve atrophy, hearing loss, and facial palsy were reported in two individuals. A mild developmental delay was reported in three affected individuals. None of the patients have torus palatinus, increased rate of fractures, osteomyelitis, hepatosplenomegaly, or pancytopenia. A missense mutation 640G→A (A214T) in the low-density lipoprotein receptor-related protein 5 (LRP5) gene was found in all affected individuals analyzed, including cases in whom craniosynostosis, a mild developmental delay, and/or macrocephaly is observed. To our knowledge, this is the first report in the literature of patients presenting with autosomal dominant osteosclerosis in whom a variable expression of craniosynostosis, macrocephaly, and mild developmental delay is observed, which is most likely associated with a mutation in the LRP5 gene. These phenotypes can therefore be added to the clinical spectrum of LRP5-associated bone disorders.
Predictive Value of BMD for Hip and Other Fractures
Authors: Johnell Olof , Kanis John A , Oden Anders , Johansson Helena , De Laet Chris , Delmas Pierre , Eisman John A , Fujiwara Seiko , Kroger Heikki , Mellstrom Dan , Meunier Pierre J , Melton L Joseph , O'Neill Terry , Pols Huibert , Reeve Jonathan , Silman Alan , Tenenhouse Alan
Published: April 2006

The relationship between BMD and fracture risk was estimated in a meta-analysis of data from 12 cohort studies of ∼39,000 men and women. Low hip BMD was an important predictor of fracture risk. The prediction of hip fracture with hip BMD also depended on age and z score.

Introduction: The aim of this study was to quantify the relationship between BMD and fracture risk and examine the effect of age, sex, time since measurement, and initial BMD value.

Materials and Methods: We studied 9891 men and 29,082 women from 12 cohorts comprising EVOS/EPOS, EPIDOS, OFELY, CaMos, Rochester, Sheffield, Rotterdam, Kuopio, DOES, Hiroshima, and 2 cohorts from Gothenburg. Cohorts were followed for up to 16.3 years and a total of 168,366 person-years. The effect of BMD on fracture risk was examined using a Poisson model in each cohort and each sex separately. Results of the different studies were then merged using weighted coefficients.

Results: BMD measurement at the femoral neck with DXA was a strong predictor of hip fractures both in men and women with a similar predictive ability. At the age of 65 years, risk ratio increased by 2.94 (95% CI = 2.02-4.27) in men and by 2.88 (95% CI = 2.31-3.59) in women for each SD decrease in BMD. However, the effect was dependent on age, with a significantly higher gradient of risk at age 50 years than at age 80 years. Although the gradient of hip fracture risk decreased with age, the absolute risk still rose markedly with age. For any fracture and for any osteoporotic fracture, the gradient of risk was lower than for hip fractures. At the age of 65 years, the risk of osteoporotic fractures increased in men by 1.41 per SD decrease in BMD (95% CI = 1.33-1.51) and in women by 1.38 per SD (95% CI = 1.28-1.48). In contrast with hip fracture risk, the gradient of risk increased with age. For the prediction of any osteoporotic fracture (and any fracture), there was a higher gradient of risk the lower the BMD. At a z score of -4 SD, the risk gradient was 2.10 per SD (95% CI = 1.63-2.71) and at a z score of -1 SD, the risk was 1.73 per SD (95% CI = 1.59-1.89) in men and women combined. A similar but less pronounced and nonsignificant effect was observed for hip fractures. Data for ultrasound and peripheral measurements were available from three cohorts. The predictive ability of these devices was somewhat less than that of DXA measurements at the femoral neck by age, sex, and BMD value.

Conclusions: We conclude that BMD is a risk factor for fracture of substantial importance and is similar in both sexes. Its validation on an international basis permits its use in case finding strategies. Its use should, however, take account of the variations in predictive value with age and BMD.
Fibrodysplasia Ossificans Progressiva (FOP), a Disorder of Ectopic Osteogenesis, Misregulates Cell Surface Expression and Trafficking of BMPRIA
Authors: de la Peña Lourdes Serrano , Billings Paul C , Fiori Jennifer L , Ahn Jaimo , Kaplan Frederick S , Shore Eileen M
Published: April 2006

FOP is a disorder in which skeletal muscle is progressively replaced with bone. FOP lymphocytes, a model system for exploring the BMP pathway in these patients, exhibit a defect in BMPRIA internalization and increased activation of downstream signaling, suggesting that altered BMP receptor trafficking underlies ectopic bone formation in this disease.

Introduction: Fibrodysplasia ossificans progressiva (FOP) is a severely disabling disorder characterized by progressive heterotopic ossification of connective tissues. Whereas the genetic defect and pathophysiology of this condition remain enigmatic, BMP4 mRNA and protein are overexpressed, and mRNAs for a subset of secreted BMP antagonists are not synthesized at appropriate levels in cultured lymphocytes from FOP patients. These data suggest involvement of altered BMP signaling in the disease. In this study, we investigate whether the abnormality is associated with defective BMP receptor function in lymphocytes.

Materials and Methods: Cell surface proteins were quantified by fluorescence-activated cell sorting (FACS). Protein phosphorylation was assayed by immunoprecipitation and immunoblotting. Protein synthesis and degradation were examined by [35S]methionine labeling and pulse-chase assays. mRNA was detected by RT-PCR.

Results: FOP lymphocytes expressed 6-fold higher levels of BMP receptor type IA (BMPRIA) on the cell surface compared with control cells and displayed a marked reduction in ligand-stimulated internalization and degradation of BMPRIA. Moreover, in control cells, BMP4 treatment increased BMPRIA phosphorylation, whereas BMPRIA showed ligand-insensitive constitutive phosphorylation in FOP cells. Our data additionally support that the p38 mitogen-activated protein kinase (MAPK) signaling pathway is a major BMP signaling pathway in these cell lines and that expression of inhibitor of DNA binding and differentiation 1 (ID-1), a transcriptional target of BMP signaling, is enhanced in FOP cells.

Conclusions: These data extend our previous observations of misregulated BMP4 signaling in FOP lymphocytes and show that cell surface overabundance and constitutive phosphorylation of BMPRIA are associated with a defect in receptor internalization. Altered BMP receptor trafficking may play a significant role in FOP pathogenesis.