bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytokines, Osteoprotegerin, and RANKL In Vitro and Histomorphometric Indices of Bone Turnover in Patients With Different Bone Diseases
Authors: Siggelkow Heide , Eidner Thorsten , Lehmann Gabriele , Viereck Volker , Raddatz Dirk , Munzel Ullrich , Hein Gert , Hüfner Michael
Published: April 2006

Cytokines are supposed to play an essential role in the regulation of the bone metabolic unit. However, information on cytokine production of primary human osteoblasts from patients with metabolic bone disease is scarce, and few attempts have been made to correlate such data to histomorphometric parameters of individual patients. We investigated 11 patients with metabolic bone disease referred to our outpatient department for bone biopsy and analyzed interleukin (IL)-1, IL-6, and TNF-α protein release and gene expression in primary osteoblast cultures. Compared with four controls, five patients showed normal cytokine protein release, whereas six patients showed much higher levels of interleukin-6 (26-fold) and TNF-α (84-fold). All three cytokines were strongly correlated concerning gene expression and/or protein levels (r = 0.72–0.96). Histomorphometric analysis of the bone samples showed that eroded surface (ES/BS) as a parameter of bone resorption was significantly associated with TNF-α. In addition, RANKL gene expression was positively associated with ES/BS and osteoclast surface (Oc.S/BS). Finally, the formation parameters osteoid volume and osteoid surface were negatively associated with TNF-α. In conclusion, in an in vitro-ex vivo model of bone cells obtained from a group of 11 patients with different forms of metabolic bone disease, cytokine release in conditioned medium was significantly associated with bone resorption and bone formation, as quantified by histomorphometry. TNF-α seemed to be the more important cytokine; its effect on bone resorption could be mediated by RANKL.
The Bone Gain Induced by Exercise in Puberty Is Not Preserved Through a Virtually Life-Long Deconditioning: A Randomized Controlled Experimental Study in Male Rats
Authors: Pajamäki Ilari , Kannus Pekka , Vuohelainen Tuomo , Sievänen Harri , Tuukkanen Juha , Järvinen Markku , Järvinen Teppo LN
Published: April 2006

To investigate the controversial issue whether exercise-induced positive effects on bone can be maintained after cessation of exercise, 100 5-week-old male Sprague-Dawley rats were used to assess the effects of long-term exercise (EX, treadmill running) and subsequent deconditioning (DC, free cage activity) on the femoral neck and femoral midshaft. At entry, the rats were randomly assigned into eight groups: four control groups (C14, C28, C42, and C56), and four exercise groups (EX, EX + DC14, EX + DC28, and EX + DC42). Rats in the exercise groups were first subjected to a 14-week period of progressively intensifying running, after which the rats of group EX were killed and the remaining exercise groups (EX + DC14, EX + DC28, and EX + DC42) were allowed to move freely in their cages for a subsequent deconditioning period of 14, 28, or 42 weeks, whereas control rats were kept free in their cages for the entire study period (0–56 weeks) and killed with their respective exercise group. At each time point, a comprehensive analysis of the femoral neck and midshaft characteristics (peripheral quantitative computed tomography analysis and fracture load [Fmax]) was performed. In comparison with their age-matched controls, 14 weeks of treadmill training resulted in significant (p < 0.05) increases in all measured femoral neck parameters of the growing male rats (i.e., +25% in total cross-sectional area [tCSA], +28% in total bone mineral content [tBMC], +11% in total bone mineral density [tBMD], and +30% in Fmax). On the contrary, no exercise-induced positive effects were seen in femoral midshaft. The exercise-induced benefits in the femoral neck were partially maintained during the deconditioning period of 14 weeks, the tCSA being +17%, tBMC +18% (both p < 0.05), and the Fmax +11% (p = 0.066) higher in the exercised group than control group. However, after 42 weeks of deconditioning, these benefits were eventually lost. In conclusion, exercise through the period of the fastest skeletal growth results in significant improvements in size, mineral mass, and strength of the femoral neck of male rats. However, these exercise-induced bone benefits are eventually lost if exercise is completely ceased, and thus, continued training is probably needed to maintain the positive effects of youth exercise into adulthood. Further studies should focus on assessing the minimal level of activity needed to maintain the exercise-induced bone gains.
Influence of Environmental Cadmium Exposure on Forearm Bone Density
Authors: Wang Hongfu , Zhu Guoying , Shi Yongxin , Weng Shifang , Jin Taiyi , Kong Qinghu , Nordberg Gunnar F
Published: April 2006

Cadmium may have both direct and indirect effects on bone turnover. It is nephrotoxic and can interfere with vitamin D metabolism. Such perturbation may result in osteoporosis and osteomalacia. In this study, a total of 790 persons (302 males and 488 females) participated; they were all over 35 years old and resided in an area near a cadmium smelter in southeast China. All participants completed a questionnaire, and bone mineral density was measured by SPA-4 single-photon absorptiometry at the radius and ulna. Cadmium content of urine was determined by graphite-furnace atomic absorption spectrophotometry as a measure of dose. The decline in bone mineral density with age in a heavily polluted area was greater than that in a control area for subjects over 60 years of age of both sexes (p < 0.05). In single regression, forearm bone densities were negatively correlated with urinary cadmium excretion in both males and females (p < 0.001), whereas stepwise regression showed that forearm bone density decreased linearly with age (p < 0.001) and urinary cadmium (p < 0.01) in both sexes, suggesting a dose-effect relationship between cadmium dose and bone mineral density. Based on the World Health Organization criteria, (bone mineral density < −2.5 SDs below the normal young adult), the prevalence of osteoporosis in women increased from 34.0% in the control area to 51.9% in the heavily polluted area (p < 0.01) among subjects over 50 years old, and the odds ratio value was 2.09 (95% CI: 1.08–4.03) for the highly polluted area compared with the control area. A striking observation in the study was the marked increase of the prevalence of fracture in the cadmium-polluted area in both sexes. It was concluded that environmental exposure to cadmium is associated with an increased loss of bone mineral density in both males and females, leading to osteoporosis and increased risk of fractures, especially in the elderly and in females.
Tail Suspension Induces Bone Loss in Skeletally Mature Mice in the C57BL/6J Strain but Not in the C3H/HeJ Strain
Authors: Amblard Daniel , Lafage-Proust Marie-Hélène , Laib Andres , Thomas Thierry , Rüegsegger Peter , Alexandre Christian , Vico Laurence
Published: April 2006

We assessed the effects of tail-suspension in two skeletal genetic backgrounds, the high C3H/HeJ (C3H) and low C57BL/6J (B6) bone masses inbred mice (male, 4-months old). Cancellous bone mass and structural parameters were evaluated in distal femoral metaphysis by three dimensional microcomputed tomography. Bone cellular activities were evaluated by histomorphometry and measurements of alkaline phosphatase activity (ALP) and osteocalcin in blood and deoxypyridinoline (D-pyr) in urine. In C3H mice, 2- and 3-week unloading experiments were performed. After an early and transient decrease in body weight, a 2-week suspension period resulted in stimulation of both bone formation rate by 45% and active osteoclastic surfaces by 19%. D-pyr did not change, but ALP and osteocalcin levels increased by 18% and 72%, respectively, in 2-week suspended mice, and osteocalcin remained elevated by 30% in the 3-week suspended mice. Such cellular modifications allowed the C3H mice to maintain their initial bone mass and trabecular structural parameters even after a 3-week suspension period. In B6 mice, 1- and 2-week unloading experiments were performed. Tail suspension resulted in decreased body weight during the first days followed by an incomplete recovery during the second week of unloading. The resorption activity was unaffected by any suspension time period, whereas a decrease of 42.5% in bone formation rate and of 21.5% in ALP were seen by the end of the first week of suspension, both values being restored after a 2-week suspension period. At this latter time, trabeculae were thinner, leading to a 24.5% cancellous bone loss. Trabecular number and connectivity, rod-plate index, and degree of anisotropy were not modified. We concluded that C3H mice constituted a unique model in which genetic background overwhelmed the usual effects of reduced biomechanical usage in bone, whereas B6 mice, compared with the standardized rat model, offered an alternative model of bone loss in a mature skeleton.
Molecular Imaging of the Skeleton: Quantitative Real-Time Bioluminescence Monitoring Gene Expression in Bone Repair and Development
Authors: Bar Iris , Zilberman Yoram , Zeira Eveline , Galun Eithan , Honigman Alik , Turgeman Gadi , Clemens Thomas , Gazit Zulma , Gazit Dan
Published: April 2006

Monitoring gene expression in vivo, noninvasively, is a critical issue in effective gene therapy systems. To date, there are no adequate molecular imaging techniques, which quantitatively monitor gene expression in vivo in skeletal development and repair. The aim of this study was to monitor gene expression in skeletal development and repair, using a real-time molecular imaging system, which quantitatively and noninvasively detects bioluminescence in vivo. Our experimental model consisted of transgenic mice harboring the luciferase marker gene under the regulation of the human osteocalcin (hOC) promoter. A new light detection cooled charge coupled device (CCCD) camera was applied to monitor luciferase expression. In vitro, mesenchymal stem cells (MSCs) isolated from bone marrow of transgenic mice exhibited hOC promoter regulation, detected by luciferase expression that correlated with their osteogenic differentiation. During development from 1 week to 1.5 years, transgenic mice exhibited transgene expression in a wide spectrum of skeletal organs, including calvaria, vertebra, tail, and limbs, reaching a peak at 1 week in most of the skeletal organs. In two skeletal repair models, bone fracture and marrow ablation, the noninvasive CCCD system revealed a peak of luciferase expression at 6 days postsurgery. All quantitative, noninvasive, real-time CCCD measurements correlated with a luciferase biochemical assay and luciferase immunohistochemistry, which demonstrated luciferase expression in hypertrophic chondrocytes and trabecular osteoblasts. Our studies show for the first time (1) the CCCD detection system is a reliable quantitative gene detection tool for the skeleton in vivo, (2) expression of luciferase regulated by the hOC promoter is significantly decreased with age in most skeletal sites, and (3) the dynamics of hOC regulation during mice skeletal development and repair in real time, quantitatively and noninvasively.
Phenotypic Characterization of Early Onset Paget's Disease of Bone Caused by a 27-bp Duplication in the TNFRSF11A Gene
Authors: Nakatsuka Kiyoshi , Nishizawa Yoshiki , Ralston Stuart H
Published: April 2006

Three different insertion mutations in the TNFRSF11A gene affecting the signal peptide of RANK have been described. An 18-bp duplication at position 84 (84dup18) is associated with the clinical syndrome of familial expansile osteolysis (FEO), whereas a 15-bp duplication at the same site (84dup15) causes the syndrome of expansile skeletal hyperphosphatasia (ESH). Here we report the phenotype of patients harboring a 27-bp duplication at position 75 (75dup27) in RANK. Affected individuals had hearing impairment and tooth loss beginning in the second or third decade. Radiographs of affected bones showed lytic and sclerotic lesions with bony enlargement and deformity. Serum alkaline phosphatase levels were elevated between 2 and 17 times above the normal range. Most patients had pelvic and skull involvement, and all had involvement of the mandible and maxilla. Most patients also had bony enlargement of the small joints of the hands, and one developed hypercalcemia during a period of immobilization. We conclude that the 75dup27 mutation of RANK causes a Paget's disease of bone-like phenotype that is distinct from, but which overlaps with, FEO and ESH. A particularly striking feature was involvement of the mandible and maxilla, but it remains to be seen if this is a specific feature of the 75dup27 mutation until further kindreds with this mutation are reported.
PTHrP Rescues ATDC5 Cells From Apoptosis Induced by FGF Receptor 3 Mutation
Authors: Yamanaka Yoshitaka , Tanaka Hiroyuki , Koike Mio , Nishimura Riko , Seino Yoshiki
Published: April 2006

An activation mutation in the FGFR3 gene causes ACH. The effects of the FGFR3 mutants on apoptosis were analyzed in a chondrogenic cell line. ACH chondrocytes exhibited marked apoptotic with downregulation of PTHrP expression. Rescue of these cells by PTHrP replacement implies a potential therapy for this disorder.

Introduction: Achondroplasia (ACH), the most common form of short-limb dwarfism, and its related disorders are caused by constitutively activated point-mutated FGFR3. Recent studies have provided a large body of evidence on chondrocyte proliferation and differentiation in these disorders. However, little is known about the possible effects of the FGFR3 mutants on apoptosis of chondrocytes.

Methods: The mutant FGFR3 genes causing ACH and thanatophoric dysplasia (TD), which is a more severe neonatal lethal form, were introduced into a chondrogenic cell line, ATDC5. Analysis of apoptosis was estimated by TUNEL assay, DNA laddering, and fluorescent measurement of mitochondrial membrane potential. Expression levels of parathyroid hormone-related peptide (PTHrP) and apoptosis-related genes were analyzed by Northern blot or immunoblot.

Results: The introduction of these mutated FGFR3s into ATDC5 cells downregulated PTHrP expression and induced apoptosis with reduction of Bcl-2 expression. Importantly, replacement of PTHrP prevented the apoptotic changes and reduction of Bcl-2 expression in ATDC5 cells expressing the ACH mutant. In parallel with the severity of disease and the activity of FGFR3, ATDC5 cells expressing TD-mutant FGFR3 showed less expression of PTHrP and Bcl-2 and induced more remarkable apoptotic changes compared with ACH-mutant expressing cells. Furthermore, overexpression of Bcl-2 inhibited apoptotic changes, suggesting that the mutant FGFR3 caused apoptosis, at least in part, through reduction of Bcl-2 expression, which seems to be downstream of PTHrP.

Conclusions: Our data suggest that excessive activation of signaling cascades mediated by the FGFR3 mutants inhibits the expression of PTHrP and Bcl-2, resulting in apoptosis of chondrocytes, possibly leading to short-limb dwarfism. Rescue of these cells by PTHrP replacement implies a potential therapy for this disorder.
Stromal Cell-Derived Factor-1 (SDF-1) Recruits Osteoclast Precursors by Inducing Chemotaxis, Matrix Metalloproteinase-9 (MMP-9) Activity, and Collagen Transmigration
Authors: Yu Xuefeng , Huang Yuefang , Collin-Osdoby Patricia , Osdoby Philip
Published: April 2006

Signals targeting OCs to bone and resorption sites are not well characterized. A chemoattractant receptor (CXCR4), highly expressed in murine OC precursors, mediated their chemokine (SDF-1)-induced chemoattraction, collagen transmigration, and MMP-9 expression. Thus, bone vascular and stromal SDF-1 may direct OC precursors into bone and marrow sites for development and bone resorption.

Introduction: Although chemokines are essential for trafficking and homing of circulating hematopoietic cells under normal and pathological conditions, their potential roles in osteoclast (OC) recruitment or function are generally unknown. CXCR4 and its unique ligand, stromal cell-derived factor-1 (SDF-1), critically control the matrix metalloproteinase (MMP)-dependent targeting of hematopoietic cells into bone and within the marrow microenvironment. Therefore, SDF-1/CXCR4 may regulate OC precursor recruitment to sites for development and activation.

Methods: Chemokine receptor mRNA expression was analyzed during OC formation induced by RANKL in murine RAW 264.7 cells. SDF-1 versus RANKL effects on chemotaxis, transcollagen migration, MMP-9 expression and activity, OC development, and bone resorption were evaluated in RAW cells or RAW-OCs.

Results: CXCR4 was highly expressed in RAW cells and downregulated during their RANKL development into bone-resorptive RAW-OCs. SDF-1, but not RANKL, elicited RAW cell chemotaxis. Conversely, RANKL, but not SDF-1, promoted RAW-OC development, TRAP activity, cathepsin K expression, and bone pit resorption, and SDF-1 did not modify these RANKL responses. Both SDF-1 and RANKL increased MMP-9, a matrix-degrading enzyme essential for OC precursor migration into developing bone marrow cavities, and increased transcollagen migration of RAW cells in a MMP-dependent manner. SDF-1 also upregulated MMP-9 in various primary murine OC precursor cells. Because RANKL induced a higher, more sustained expression of MMP-9 in RAW cells than did SDF-1, MMP-9 may have an additional role in mature OCs. Consistent with this, MMP-9 upregulation during RANKL-induced RAW-OC development was necessary for initiation of bone pit resorption.

Conclusions: SDF-1, a chemokine highly expressed by bone vascular endothelial and marrow stromal cells, may be a key signal for the selective attraction of circulating OC precursors into bone and their migration within marrow to appropriate perivascular stromal sites for RANKL differentiation into resorptive OCs. Thus, SDF-1 and RANKL likely serve complementary physiological functions, partly mediated through increases in MMP-9, to coordinate stages of OC precursor recruitment, development, and function.
Expression of Chicken 75-kDa Gelatinase B-like Enzyme in Perivascular Chondrocytes Suggests Its Role in Vascularization of the Growth Plate
Authors: Tong A , Reich A , Genin O , Pines M , Monsonego-Ornan E
Published: April 2006

A newly cloned avian 75-kDa gelatinase B-like enzyme is expressed by the cells surrounding the blood vessels of the growth plate and upregulated by angiogenic substances in cultured chondrocytes. Despite its low homology to mammalian gelatinase-B, the avian 75-kDa seems to function similarly in the context of endochondral bone formation.

Introduction: Gelatinase B/metalloproteinase (MMP)-9, a zinc-dependent protease of the MMP family, is a key regulator in the final step of endochondral ossification, Recently an avian 75-kDa gelatinase B-like enzyme that shows low sequence similarity to the mammalian enzyme (59% on the protein level) was cloned and characterized. However, its expression pattern in the chicken growth plate and its role in bone formation have not, so far, been examined.

Results: Based on the published sequence, we cloned a 700-bp fragment from cDNA of the chicken growth plate and studied its expression pattern in primary chondrocytes. Because the basal expression level of gelatinase B was almost undetectable, we induced its expression by different culturing conditions, the most dramatic induction achieved by treatment with retinoic acid, which is known as an inducer of vascular invasion in the epiphyseal plates. The gelatinolitic activity, checked by zymography, detected bands corresponding to the gelatinase A and B as well as a new high-molecular weight band of ∼200 kDa. We further studied the expression pattern of gelatinase B by in situ hybridization. The gelatinase B was expressed by the cells surrounding the blood vessels penetrating the growth plate and by chondrocytes located in the front of these vascular invasions in the borders between the bone and the cartilage, resembling the expression of mouse gelatinase B in the growth plate. The induction of rickets by a vitamin D-deficient diet reduced the expression levels of gelatinase B in the growth plate of 12-day-old chickens but did not affect the expression of gelatinase A mRNA.

Conclusion: The chicken growth plate has a distinctly different structure from the mammalian one: it is much wider, it contains more cells in each zone, and the blood vessels penetrate deeper into the hypertrophic zone. Nevertheless, the upregulation of the avian 75-kDa gelatinase B-like enzyme by vitamins A and D, coupled with its perivascular expression pattern in the growth plate, implies a similar role for the mammalian and avian genes in bone formation.
Direct Suppressive Effect of Acute Metabolic and Respiratory Alkalosis on Parathyroid Hormone Secretion in the Dog
Authors: Lopez Ignacio , Rodriguez Mariano , Felsenfeld Arnold J , Estepa Jose Carlos , Aguilera-Tejero Escolastico
Published: April 2006

Acute alkalosis may directly affect PTH secretion. The effect of acute metabolic and respiratory alkalosis was studied in 20 dogs. PTH values were lower in the metabolic (5.6 ± 0.8 pg/ml) and respiratory (1.8 ± 0.6 pg/ml) alkalosis groups than in the control group (27 ± 5 pg/ml). Acute alkalosis is an independent factor that decreases PTH values during normocalcemia and delays the PTH response to hypocalcemia.

Introduction: We recently showed that acute metabolic and respiratory acidosis stimulated PTH secretion. This study was designed to evaluate whether acute metabolic and respiratory alkalosis suppressed parathyroid hormone (PTH) secretion.

Materials and Methods: Three groups of 10 dogs were studied: control, acute metabolic alkalosis, and acute respiratory alkalosis. Metabolic alkalosis was induced with an infusion of sodium bicarbonate and respiratory alkalosis by hyperventilation. Calcium chloride was infused to prevent alkalosis-induced hypocalcemia during the first 60 minutes. During the next 30 minutes, disodium EDTA was infused to induce hypocalcemia and to evaluate the PTH response to hypocalcemia. Because the infusion of sodium bicarbonate resulted in hypernatremia, the effect of hypernatremia was studied in an additional group that received hypertonic saline.

Results: After 60 minutes of a normocalcemic clamp, PTH values were less (p < 0.05) in the metabolic (5.6 ± 0.8 pg/ml) and respiratory (1.8 ± 0.6 pg/ml) alkalosis groups than in the control group (27 ± 5 pg/ml); the respective blood pH values were 7.61 ± 0.01, 7.59 ± 0.02, and 7.39 ± 0.02. The maximal PTH response to hypocalcemia was similar among the three groups. However, the maximal PTH response was observed after a decrease in ionized calcium of 0.20 mM in the control group but not until a decrease of 0.40 mM in the metabolic and respiratory alkalosis groups. In contrast to the metabolic alkalosis group, hypernatremia (157 ± 2 mEq/liter) in the hypertonic saline group was associated with an increased PTH value (46 ± 4 pg/ml). Finally, the half-life of intact PTH was not different among the control and two alkalosis groups.

Conclusions: Acute metabolic and respiratory alkalosis markedly decreased PTH values during normocalcemia and delayed the PTH response to hypocalcemia. Whether acute metabolic and respiratory alkalosis affect PTH and calcium metabolism in such settings as the postprandial alkaline tide (metabolic alkalosis) and acute sepsis (respiratory alkalosis) deserves to be evaluated in future studies.
Dentin Matrix Protein 1 Is Expressed in Human Lung Cancer
Authors: Chaplet M , De Leval L , Waltregny D , Detry C , Fornaciari G , Bevilacqua G , Fisher LW , Castronovo V , Bellahcène A
Published: April 2006

We have previously shown that breast and prostate cancers express bone matrix proteins. DMP1 expression was evaluated in 59 human lung cancer samples at the protein and mRNA levels. It was detectable in 80% of the cases, suggesting a potential role for DMP1 in tumor progression and bone metastasis.

Introduction: Previously, we and others have shown that bone extracellular matrix proteins such as bone sialoprotein (BSP) and osteopontin (OPN) are expressed in various types of cancer that are characterized by a high affinity for bone including breast, prostate, and lung adenocarcinoma. Based on biochemical and genetic features, BSP, OPN, dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP) have been recently classified in a unique family named SIBLING (small integrin-binding ligand, N-linked glycoprotein). Therefore, we investigated whether DMP1 could also be detected in osteotropic cancers.

Materials and Methods: We first used a cancer array for evaluating the relative abundance of DMP1 transcript in a broad spectrum of human cancer tissues. This screening showed that DMP1 was strongly detectable in lung tumors compared with normal corresponding tissue. In a second step, we used an immunophosphatase technique and a specific polyclonal antibody directed against DMP1 to examine the expression of DMP1 in 59 human non-small cell lung cancer samples, including 29 squamous carcinoma, 20 adenocarcinoma, and 10 bronchioloalveolar carcinoma. Student's t-test was used to determine the statistical significance of immunostaining scores between the lung cancer histological groups studied and between cancer and normal lung tissues.

Results: Our results show that DMP1 is detectable in 90% of the adenocarcinoma and squamous carcinoma analyzed while 8 of 10 bronchioloalveolar specimens were negative. DMP1 immunostaining intensity and extent scores were significantly higher in adenocarcinoma (p = 0.0004) and squamous carcinoma (p < 0.0001) samples compared with adjacent normal lung tissue. In situ hybridization experiments confirmed that DMP1 mRNA is localized in lung cancer cells.

Conclusion: In this study, we show that a third SIBLING protein is ectopically expressed in lung cancer. The role of DMP1 in lung cancer is largely unknown. Further studies are required to determine the implication of this protein, next to its sisters SIBLING proteins, in tumor progression and bone metastasis development.
Chloride Channel 7 (ClCN7) Gene Mutations and Autosomal Dominant Osteopetrosis, Type II
Authors: Waguespack Steven G , Koller Daniel L , White Kenneth E , Fishburn Tonya , Carn Gwenaelle , Buckwalter Kenneth A , Johnson Michelle , Kocisko Maureen , Evans Wayne E , Foroud Tatiana , Econs Michael J
Published: April 2006

ADO2 is an uncommon sclerosing bone disorder with incomplete penetrance and variable expressivity. Positional candidate studies were performed to identify the gene responsible for ADO2. In 11 of 12 kindreds, five different missense mutations were identified in the ClCN7 gene, indicating the genetic basis and possible dominant negative mechanism for ADO2.

Introduction: Autosomal dominant osteopetrosis, type II (ADO2) is an uncommon sclerosing bone disorder with a distinct radiographic appearance and unique clinical characteristics. We present the results from our genetic studies designed to identify the ADO2 gene through a positional candidate approach.

Methods: Having identified 12 families with ADO2, we initially performed linkage studies in our seven largest kindreds and observed a summed maximum LOD score of 15.91 at marker D16S521 on chromosome 16p13.3. Critical meiotic recombination events further narrowed the putative gene region to a 7.6-cM area, which contains the candidate genes ATP6L and chloride channel 7 (ClCN7). We screened affected individuals from each ADO2 family for mutations in these genes using direct sequencing. Identified mutations were subsequently confirmed through direct sequencing or restriction fragment length polymorphism analysis. We then calculated the overall disease penetrance rate after all available at-risk family members were assessed for ClCN7 gene mutations.

Results: No ATP6L mutations were identified in affected subjects. Subsequently, as ClCN7 gene mutations were being reported, we identified two novel (L213F, R762L) and three known (G215R, R286W, R767W) missense mutations in 11 kindreds. In our large sample, disease penetrance was 66% (62 clinically affected individuals/94 subjects with the gene mutation). To date, nine different mutations have been discovered in the ClCN7 gene in 22 of 23 ADO2 families studied.

Conclusions:We conclude that mutations in the ClCN7 gene are responsible for ADO2 and that genetic heterogeneity is unlikely to exist in this disorder. Based on the preponderance of missense mutations and the knowledge that chloride channels probably function as dimers, it seems that heterozygous ClCN7 gene mutations may cause ADO2 through a dominant negative mechanism.
Analysis of Environmental Factors in Familial Versus Sporadic Paget's Disease of Bone—The New England Registry for Paget's Disease of Bone
Authors: Seton Margaret , Choi Hyon K , Hansen Marc F , Sebaldt Rolf J , Cooper Cyrus
Published: April 2006

A registry for Paget's Disease (PD) was created to study the environmental and familial features of this disorder. This study examines the first 202 people enrolled. Those with a family history of PD (20%) suffered earlier deformity and fracture, and tended to have grandparents born abroad. These findings suggest heritage is important in PD; the role of environment remains unclear.

Introduction: The New England Registry for Paget's Disease of Bone is a database that was created to explore the distribution and determinants of disease frequency.

Methods: Using a case-series design, we explored the association of environmental factors in expression of the disease, comparing those patients with familial Paget's disease to those with sporadic Paget's disease (PD).

Results and Conclusions: Analysis of the first 202 patients enrolled in the registry revealed a positive family history (FH) in 41 (20%). Significant findings in this cohort included an earlier age of onset (51 years FH+ versus 59 years FH−, p < 0.05), a trend to a higher incidence of bone deformity (49% versus 33%, p < 0.1), and an increased fracture rate (27% versus 11%, p < 0.05). Persons with a FH of PD were less likely to record the United States or Canada as their grandparents' birthplace (p < 0.01), and sibships tended to be larger (p < 0.05). A history of measles infection, childhood exposure to pets, milk ingestion, year of immigration to the United States, birth order, level of education, and functional status did not distinguish the two groups. The database confirms the high prevalence of familial PD and supports the theory that heritable factors are important in the pathogenesis of this focal disorder of bone metabolism.
Site and Gender Specificity of Inheritance of Bone Mineral Density
Authors: Duncan Emma L , Cardon Lon R , Sinsheimer Janet S , Wass John AH , Brown Matthew A
Published: April 2006

Differences in genetic control of BMD by skeletal sites and genders were examined by complex segregation analysis in 816 members of 147 families with probands with extreme low BMD. Spine BMD correlated more strongly in male-male comparisons and hip BMD in female-female comparisons, consistent with gender- and site-specificity of BMD heritability.

Introduction: Evidence from studies in animals and humans suggests that the genetic control of bone mineral density (BMD) may differ at different skeletal sites and between genders. This question has important implications for the design and interpretation of genetic studies of osteoporosis.

Methods: We examined the genetic profile of 147 families with 816 individuals recruited through probands with extreme low BMD (T-score < −2.5, Z-score < −2.0). Complex segregation analysis was performed using the Pedigree Analysis Package. BMD was measured by DXA at both lumbar spine (L1-L4) and femoral neck.

Results: Complex segregation analysis excluded purely monogenic and environmental models of segregation of lumbar spine and femoral neck BMD in these families. Pure polygenic models were excluded at the lumbar spine when menopausal status was considered as a covariate, but not at the femoral neck. Mendelian models with a residual polygenic component were not excluded. These models were consistent with the presence of a rare Mendelian genotype of prevalence 3–19 %, causing high BMD at the hip and spine in these families, with additional polygenic effects. Total heritability range at the lumbar spine was 61–67 % and at the femoral neck was 44–67 %. Significant differences in correlation of femoral neck and lumbar spine BMD were observed between male and female relative pairs, with male-male comparisons exhibiting stronger lumbar spine BMD correlation than femoral neck, and female-female comparisons having greater femoral neck BMD correlation than lumbar spine. These findings remained true for parent-offspring correlations when menopausal status was taken into account. The recurrence risk ratio for siblings of probands of a Z-score < −2.0 was 5.4 at the lumbar spine and 5.9 at the femoral neck.

Conclusions: These findings support gender- and site-specificity of the inheritance of BMD. These results should be considered in the design and interpretation of genetic studies of osteoporosis.
Fracture Prediction From Bone Mineral Density in Japanese Men and Women
Authors: Fujiwara Saeko , Kasagi Fumiyoshi , Masunari Naomi , Naito Kumiko , Suzuki Gen , Fukunaga Masao
Published: April 2006

In a cohort of 2356 Japanese elderly, after adjusting for age and prevalent vertebral fracture, baseline BMD predicted the risk of spine and hip fracture with similar RR to that obtained from previous reports in whites. The RR per SD decrease in BMD for fracture declined with age.

Introduction: Low bone mineral density (BMD) is one of the most important predictors of a future fracture. However, we are not aware of any reports among Japanese in Japan.

Materials and Methods: We examined the association of BMD with risk of fracture of the spine or hip among a cohort of 2356 men and women aged 47–95 years, who were followed up by biennial health examinations. Follow-up averaged 4 years after baseline measurements of BMD that were taken with the use of DXA. Vertebral fracture was assessed using semiquantitative methods, and the diagnosis of hip fracture was based on medical records. Poisson and Cox regression analysis were used.

Results: The incidence was twice as high in women as in men, after adjusting for age. After adjusting for baseline BMD and prevalent vertebral fracture, however, the gender difference was no longer significant. Age, baseline BMD of spine and femoral neck, and prior vertebral fracture predicted vertebral fracture and hip fracture. Loss of absolute BMD of the femoral neck predicted spine fracture, after adjusting for baseline BMD; rates of change in percent BMD, weight, height, body mass index, and age at menopause did not. The predictive value of baseline BMD for vertebral fracture risk was similar in men and women. The relative risk (RR) for vertebral fracture and hip fracture per SD decrease in BMD declined with age, after adjustment for prevalent vertebral fractures.

Conclusions: Baseline BMD, loss of femoral neck BMD, and prior vertebral fracture predict the risk of spine and hip fracture in Japanese with similar RR to that obtained from previous reports in whites. The RR per SD decrease in BMD for fracture declined with age, suggesting that factors other than BMD might play a greater role in the elderly.
Impaired Fracture Healing in the Absence of TNF-α Signaling: The Role of TNF-α in Endochondral Cartilage Resorption
Authors: Gerstenfeld LC , Cho T-J , Kon T , Aizawa T , Tsay A , Fitch J , Barnes GL , Graves DT , Einhorn TA
Published: April 2006

TNF-α is a major inflammatory factor that is induced in response to injury, and it contributes to the normal regulatory processes of bone resorption. The role of TNF-α during fracture healing was examined in wild-type and TNF-α receptor (p55−/−/p75−/−)-deficient mice. The results show that TNF-α plays an important regulatory role in postnatal endochondral bone formation.

Introduction: TNF-α is a major inflammatory factor that is induced as part of the innate immune response to injury, and it contributes to the normal regulatory processes of bone resorption.

Methods: The role of TNF-α was examined in a model of simple closed fracture repair in wild-type and TNF-α receptor (p55−/−/p75−/−)-deficient mice. Histomorphometric measurements of the cartilage and bone and apoptotic cell counts in hypertrophic cartilage were carried out at multiple time points over 28 days of fracture healing (n = 5 animals per time point). The expression of multiple mRNAs for various cellular functions including extracellular matrix formation, bone resorption, and apoptosis were assessed (triplicate polls of mRNAs).

Results and Conclusions: In the absence of TNF-α signaling, chondrogenic differentiation was delayed by 2–4 days but subsequently proceeded at an elevated rate. Endochondral tissue resorption was delayed 2–3 weeks in the TNF-α receptor (p55−/−/p75−/−)-deficient mice compared with the wild-type animals. Functional studies of the mechanisms underlying the delay in endochondral resorption indicated that TNF-α mediated both chondrocyte apoptosis and the expression of proresorptive cytokines that control endochondral tissue remodeling by osteoclasts. While the TNF-α receptor ablated animals show no overt developmental alterations of their skeletons, the results illustrate the primary roles that TNF-α function contributes to in promoting postnatal fracture repair as well as suggest that processes of skeletal tissue development and postnatal repair are controlled in part by differing mechanisms. In summary, these results show that TNF-α participates at several functional levels, including the recruitment of mesenchymal stem, apoptosis of hypertrophic chondrocytes, and the recruitment of osteoclasts function during the postnatal endochondral repair of fracture healing.
A Missense Mutation in the Mouse Col2a1 Gene Causes Spondyloepiphyseal Dysplasia Congenita, Hearing Loss, and Retinoschisis
Authors: Donahue Leah Rae , Chang BO , Mohan Subburaman , Miyakoshi Nao , Wergedal Jon E , Baylink David J , Hawes Norman L , Rosen Clifford J , Ward-Bailey Patricia , Zheng Qing Y , Bronson Roderick T , Johnson Kenneth R , Davisson Muriel T
Published: April 2006

A missense mutation in the mouse Col2a1 gene has been discovered, resulting in a mouse phenotype with similarities to human spondyloepiphyseal dysplasia (SED) congenita. In addition, SED patients have been identified with a similar molecular mutation in human COL2A1. This mouse model offers a useful tool for molecular and biological studies of bone development and pathology.

Introduction: A new mouse autosomal recessive mutation has been discovered and named spondyloepiphyseal dysplasia congenita (gene symbol sedc).

Materials and Methods: Homozygous sedc mice can be identified at birth by their small size and shortened trunk. Adults have shortened noses, dysplastic vertebrae, femora, and tibias, plus retinoschisis and hearing loss. The mutation was mapped to Chr15, and Col2a1 was identified as a candidate gene.

Results: Sequence analyses revealed that the affected gene is Col2a1, which has a missense mutation at exon 48 causing an amino acid change of arginine to cysteine at position 1417. Two human patients with spondyloepiphyseal dysplasia (SED) congenita have been reported with the same amino acid substitution at position 789 in the human COL2A1 gene.

Conclusions: Thus, sedc/sedc mice provide a valuable model of human SED congenita with molecular and phenotypic homology. Further biochemical analyses, molecular modeling, and cell culture studies using sedc/sedc mice could provide insight into mechanisms of skeletal development dependent on Col2a1 and its role in fibril formation and cartilage template organization.
Noninvasive In Vivo Monitoring of Bone Architecture Alterations in Hindlimb-Unloaded Female Rats Using Novel Three-Dimensional Microcomputed Tomography
Authors: David Valentin , Laroche Norbert , Boudignon Benjamin , Lafage-Proust Marie-Hélène , Alexandre Christian , Ruegsegger Peter , Vico Laurence
Published: April 2006

We tested a novel microcomputed tomograph designed to longitudinally and noninvasively monitor bone alterations in hindlimb-unloaded female rats at a resolution of 26 μm over a period of 3 weeks. This prototype has a potential to detect three-dimensional trabecular microarchitectural changes induced by growth and unloading.

Introduction: Until now, data concerning structural changes of cancellous bone have only been available after necropsy of animals. In this study, we tested a novel microcomputed tomography (μCT) technique designed to monitor such changes repeatedly at a resolution of 26 μm with an acquisition time of about 10 minutes to map the entire proximal tibial metaphysis.

Materials and Methods: Four-month-old female Wistar rats were randomized to seven groups of 10 animals to be either tail-suspended or to act as controls. μCT and DXA measurements were performed at 0, 7, 14, and 23 days in suspended and control rats. One group was killed at each of these time points, and bone samples were processed for histomorphometry and ex vivo μCT.

Results: We verified that a good correlation was obtained between two-dimensional bone parameters evaluated in longitudinal tibial sections either by histomorphometry or μCT and μCT parameters obtained from either in vivo or ex vivo tibias. The longitudinal survey allowed earlier detection of both growth and unloading-related bone changes than the transverse survey. In controls, aging induced denser bones, reorganization of the trabecular network toward a more oriented plate-like structure, and an isotropic pattern. Unloading first inhibited cortical and cancellous bone growth and then induced bone loss characterized by fewer trabeculae, reduced connectivity density, and enhanced structure model index (SMI), revealing a lighter cancellous structure with development of rod-like characteristics.

Conclusion: We show for the first time that this μCT prototype has a great potential to accurately, repeatedly, reliably, and rapidly investigate alterations of three-dimensional trabecular microarchitecture.
Cdx-2 Polymorphism in the Promoter Region of the Human Vitamin D Receptor Gene Determines Susceptibility to Fracture in the Elderly
Authors: Fang Yue , Van Meurs Joyce BJ , Bergink Arjan P , Hofman Albert , Van Duijn Cornelia M , Van Leeuwen Johannes Ptm , Pols Huibert AP , Uitterlinden André G
Published: April 2006

A Cdx-2 binding site polymorphism (G to A) in the promoter region of the human vitamin D receptor gene was reported. In an ecological study in eight ethnic groups and an association study in 2848 elderly whites, we found the A-allele to be associated with decreased fracture risk. Our findings expand previous similar findings in a Japanese study to whites and show a relationship with fracture risk of this functional polymorphism.

Introduction: A single nucleotide polymorphism (SNP) within a binding site of the intestinal-specific transcription factor Cdx-2 in the promoter region of the human vitamin D receptor (VDR) gene was previously reported. It was found to modulate the transcription of the hVDR gene and to be associated with decreased bone mineral density in a small group of postmenopausal Japanese women. In this study, we investigated the relationship between the VDR Cdx-2 genotype and risk of fracture.

Methods: We first determined the location of this SNP in the VDR gene by sequencing analysis, and we developed an allele-specific multiplex polymerase chain reaction test to determine the Cdx-2 genotype. We then performed an ecological study in eight ethnic groups and an association analysis in a large epidemiological cohort of 2848 Dutch white men and women, ≥55 years old.

Results and Conclusions: The location of the G to A substitution was found in the promoter region of exon 1e (1e-G−1739A) of the VDR gene. By comparing the frequency of the A-allele in eight different ethnic groups, we observed a negative correlation between prevalence of the A-allele and published hip fracture incidence rates in these ethnic groups (p = 0.006 for men and p = 0.02 for women), suggesting a protective effect of this allele on fracture risk. Subsequently, in the association study, the A-allele (population frequency 19%) was observed to have a protective effect on occurrence of osteoporotic fractures, especially for nonvertebral fracture in women (relative risk of AA versus GG genotype is 0.2; 95% CI, 0.05–0.8). This effect remained after adjustment for age, weight, and bone mineral density. We conclude that the A-allele of the VDR Cdx-2 polymorphism is present in whites, albeit at low frequency, and show a protective effect of this allele on risk of fracture.
Reduced Iliac Cancellous Osteocyte Density in Patients With Osteoporotic Vertebral Fracture
Authors: Qiu Shijing , Rao D Sudhaker , Palnitkar Saroj , Parfitt A Michael
Published: April 2006

Iliac cancellous osteocyte density declines with age, but its relationship to vertebral fracture pathogenesis is unknown. We performed iliac bone biopsy in 44 women with clinical vertebral fracture and 56 healthy women. The fracture patients had 34% fewer osteocytes but no reduction in percent occupied lacunae. Some patients destined to sustain vertebral fracture make cancellous bone with fewer osteocytes.

Introduction: Patient's with vertebral fracture have less bone than appropriate healthy controls, but other factors may contribute to bone fragility. Iliac cancellous osteocyte density declines with age in healthy women; we asked whether this variable differed between fracture patients and healthy controls.

Methods: Two groups of women were assembled. Forty-four (mean age, 66.2 years) had unequivocal evidence of bone fragility manifested as painful nontraumatic vertebral fracture, and 56 (mean age, 62.2 years) were skeletally healthy. All subjects underwent iliac bone biopsy. From archival embedded biopsy cores, new sections were stained with Goldner's trichrome, in which we enumerated osteocyte-occupied lacunae (stained), empty lacunae (unstained), and total lacunae per bone area.

Results: Cancellous osteocyte density was 34% lower in the fracture group than in the controls (p < 0.001); this difference was not a consequence of higher turnover, having less bone, or the small difference in age. The area under the receiver operating characteristic (ROC) curve for discrimination between the groups was >90% for osteocyte density and <75% for bone volume/tissue volume (BV/TV). The disease-related osteocyte deficit was accompanied by a proportionate reduction in empty lacunae and no change in percent occupied lacunae; therefor, it was not the result of premature death. Both superficial bone (<25 μm from the surface) and deep bone (>45 μm from the surface) were affected. In contrast, the age-related deficit is accompanied by an increase in empty lacunae and fall in percent osteocyte-occupied lacunae and occurs only in deep bone, but not in superficial bone.

Conclusions: In some patients destined to sustain spontaneous vertebral compression fracture, iliac cancellous bone is made with fewer osteocytes than normal; the mechanism of osteocyte incorporation into bone needs more detailed study. Osteocyte deficiency could contribute to bone fragility, either by impairing the detection of fatigue microdamage or by reducing canalicular fluid flow. Current practices of defining vertebral fracture based on morphometry alone regardless of symptoms, and diagnosing osteoporosis based on bone densitometry alone regardless of fracture history, should be reexamined.