bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Dihydrotestosterone on Bone Biochemical Markers in Sham and Oophorectomized Rats
Authors: Mason R. A. , Morris H. A.
Published: April 2006

Evidence exists to suggest that androgens stimulate bone formation in the estrogen-deficient state, however the mechanism of action is unclear. The following study investigates the effect of dihydrotestosterone (DHT) on biochemical markers of bone turnover and calcium homeostasis in sham and oophorectomized (oophx) rats when either vehicle, 40, 80, or 160 mg/kg body weight (bw) DHT were administered at the time of operation or at 15 weeks postoperation. Serum alkaline phosphatase (ALP) increased following DHT administration in sham and oophx rats in all groups (mean ALP ± SEM [U/l] week 8; sham vehicle, 40 ± 7; sham 160 mg DHT/kg bw, 72 ± 5; oophx vehicle, 60 ± 6; oophx 160 mg DHT/kg bw, 88 ± 11) (p < 0.001). In contrast, serum osteocalcin was significantly suppressed in oophx rats administered DHT 15 weeks following operation (mean osteocalcin ± SEM [μg/l] week 8; oophx vehicle, 17.6 ± 3.5; oophx 160 mg DHT/kg bw, 10.5 ± 1) (p < 0.01). Urine deoxypyridinoline was significantly decreased when DHT was administered 15 weeks postoophorectomy (p < 0.001); however, urine hydroxyproline was not affected by DHT treatment in any group. Urine calcium was decreased by DHT treatment (mean Ca/Cr ± SEM week 8; sham vehicle, 0.87 ± 0.13; sham 160 mg DHT/kg bw, 0.24 ± 0.08; oophx vehicle, 0.68 ± 0.16; oophx 160 mg DHT/kg bw, 0.45 ± 0.1) (p < 0.005) which was associated with an increase in the renal tubular reabsorption of calcium (p < 0.05). This study demonstrates the direct effects of DHT on both bone cell activities and the renal handling of calcium.
Bone Elasticity and Ultrasound Velocity Are Affected by Subtle Changes in the Organic Matrix
Authors: Mehta Shreefal S. , Öz Orhan K. , Antich Peter P.
Published: April 2006

The mechanical competence of bone can be studied through the measurement of the components of its material elasticity, a property which can vary both in magnitude and in dependence upon orientation (anisotropy). While it is known that the elasticity is largely determined by the mineral constituents of the bone matrix, it is nonetheless clear that it must be also dependent upon the remaining constituents of bone material. In this work, the influence of organic components on the elasticity is explored by altering specific constituents of the bone matrix to varying degrees. This study addresses two questions: first, are the resulting changes in elasticity strongly or weakly dependent upon direction, and second, are they substantially dependent upon the nature and magnitude of the induced matrix alteration? To answer these questions, we performed different chemical manipulations of the bone matrix and measured the changes in elasticity and velocity using the technique of ultrasound critical angle reflectometry. Altering the properties of the organic matrix resulted in substantial and complex changes in the elasticity of bone. The observed changes were strongly dependent upon direction, could not be explained by changes in density alone, and varied strongly with the specific chemical treatment of the matrix. Immersion in urea selectively affected protein components of the organic matrix and resulted in reversible changes in velocity and elasticity, while removal of collagen caused anisotropic decreases and removal of all organic matter caused a collapse of all components of the elasticity. In conclusion, this study confirms that the organic matrix exerts a profound influence on the elasticity and indicates that the measurement of elastic properties at multiple directions is necessary in the assessment of bone mechanical competence.
Associations of Physical Activity and Calcium Intake with Bone Mass and Size in Healthy Women at Different Ages
Authors: Uusi-Rasi Kirsti , Sievänen Harri , Vuori Ilkka , Pasanen Matti , Heinonen Ari , Oja Pekka
Published: April 2006

Weight-bearing exercise and calcium intake are known to contribute to bone density. However, the relative significance of physical activity and calcium intake in the development of bone characteristics in functionally different weight-bearing and nonweight-bearing bone sites at different ages is poorly known. A total of 422 women in three age groups (25–30, 40–45, and 60–65 years) were screened from 1017 women and divided into four groups by their level of physical activity (high [PA+] and low [PA−]) and calcium intake (high [Ca+] and low [Ca−]). Total body bone mineral content (TBBMC), areal bone mineral density (BMD) of the femoral neck and distal radius, and selected dimensions and estimated strength variables (bone width, cortical wall thickness, cross-sectional moment of inertia, and section modulus of the femoral and radial shafts) were measured with dual-energy X-ray absorptiometry. Both high physical activity and high calcium intake were associated with a higher TBBMC when compared with low activity and calcium intake (1.8% and 4.6%, respectively). The BMD of the weight-bearing femoral neck was 5% higher in the PA+ groups than in the PA− groups, whereas calcium intake showed no such significant association. Neither physical activity nor calcium intake was associated with the BMD of the nonweight-bearing radius. However, both high physical activity and high calcium intake were related to larger and mechanically more competent bones in the femoral and radial shafts, the association for physical activity being stronger with increasing age. No significant interaction between physical activity and calcium intake was found with respect to any of the bone variables. These data from a cross-sectional study suggest that a moderate level of physical activity or a sufficient level of calcium intake, if maintained from childhood, can result in considerable long-term improvement in the mechanical competence of the skeleton. The clinical relevance of these findings is further emphasized by the fact that the observed patterns of physical activity and calcium intake pertain to customary lifestyle and are thus feasible targets for the primary prevention of osteoporosis.
Osteoprogenitor Cells as Targets for Ex Vivo Gene Transfer
Authors: Dr. Onyia J. E. , Clapp D. W. , Long H. , Hock J. M.
Published: April 2006

We transduced osteoprogenitor cells with recombinant retrovirus and analyzed proviral integration patterns into chromosomal DNA to detect for the first time the clonal and cellular fate of osteoprogenitor-derived progeny cells. Metaphyseal bone cells and diaphyseal stromal cells were isolated from the distal femurs of young rats, transduced with the vM5neolacZ recombinant retrovirus, and selected in the neomycin analog, G418. Following surgical marrow ablation of a femur in one leg of mature rats, retroviral-transduced metaphyseal or diaphyseal cells were injected into the ablated site. These rats were killed 5–6 days later. Metaphyseal and diaphyseal cells were isolated from distal femurs, selected in G418, and stained for β-galactosidase (β-gal+). The number and clonal origin of transduced progenitor cells were determined. High numbers of β-galactosidase colonies with an osteoblast phenotype were obtained following metaphyseal transplants and detected in 100% of metaphyseal and none of diaphyseal specimens. In contrast, β-galactosidase colonies derived from diaphyseal transplants were detected in 50% of specimens in both the metaphysis and diaphysis, and the absolute number of progenitor cell colonies was 60-fold less than metaphyseal transplants. Provirus was only detected in the ablated bones and not in the contralateral bone or other tissues. Proviral integration fragment analysis showed a single integration site for recovered metaphyseal cell clones, consistent with their origination from a common single progenitor. This is one of the first demonstrations of successful transplantation of clonal osteoprogenitors to their site of origin in bone. It may be possible to use these cells to target genes to bone for therapeutic use in skeletal and hematopoietic diseases.
Colony Stimulating Factor-1–Induced Osteoclast Spreading Depends on Substrate and Requires the Vitronectin Receptor and the c-src Proto-Oncogene
Authors: Teti Anna , Taranta Anna , Migliaccio Silvia , Degiorgi Annamaria , Santandrea Elena , Villanova Ida , Faraggiana Tullio , Chellaiah Meena , Hruska Keith A.
Published: April 2006

The colony stimulating factor 1 (CSF-1) regulates osteoclastogenesis and bone resorption. Mutations in the CSF-1 gene cause an osteopetrosis characterized by the absence of osteoclasts. Mature osteoclasts respond to CSF-1 with inhibition of bone resorption and an increment of cell spreading. Herein we demonstrate that CSF-1–induced osteoclast spreading depends on the substrate the osteoclast interacts with and requires integrity of the vitronectin receptor and of the c-src proto-oncogene. Rabbit osteoclasts were allowed to attach to glass, serum, osteopontin, and bone substrates, and were treated with 10 ng/ml human recombinant CSF-1 for 4 h. In osteoclasts plated on glass, the cytokine induced 70% inhibition of bone resorption and 1.8-fold stimulation of cell spreading, without changes in podosome expression and microfilament array. In contrast, CSF-1 induced a 2.5-fold increase of osteoclasts showing filopodia, and a 9.5-fold increase of osteoclasts presenting lamellipodia, indicating that membrane motility was required for cell spreading. Osteoclasts plated on serum substrates showed a 50% reduction of spontaneous spreading. However, in this circumstance, CSF-1 still stimulated an increase of osteoclast area. In osteoclasts cultured on osteopontin substrate or on bone slices, an inhibition of CSF-1–induced osteoclast spreading was observed. To establish involvement of the vitronectin receptor and c-src proto-oncogene, cells were treated with the αvβ3 integrin neutralizing antibody, LM609, or c-src antisense oligonucleotides, which reduced CSF-1–induced osteoclast spreading by 57% and 60%, respectively. The results demonstrate that CSF-1–induced osteoclast spreading requires both the vitronectin receptor and the c-src proto-oncogene and that this action is modulated by the adhesion substrata.
Effects of Estrogen Therapy of Postmenopausal Women on Cytokines Measured in Peripheral Blood
Authors: Rogers Angela , Eastell Richard
Published: April 2006

Estrogen replacement therapy (ERT) is known to prevent bone loss following the menopause, but the mechanism for this is unclear. Estrogen may suppress the secretion of certain bone-resorbing cytokines. The aim of this study was to assess the effect of ERT on the levels of cytokines measured in peripheral blood. We measured cytokines in 10 postmenopausal women (ages 56–59, 3–9 years since menopause) treated with ERT and 10 age-matched (54–59 years, 4–10 years since menopause) untreated women as controls. Samples of blood were taken and used for mononuclear cell cultures, whole blood (WB) cultures, and the separation of serum. The cultures were treated with lipopolysaccharide (LPS; 500 ng/ml) and hydrocortisone (10−6 M). The conditioned medium from cultures and the serum were then assayed for interleukin-6 (IL-6), IL-1α, IL-1β, IL-1 IL-1ra, tumor necrosis factor alpha (TNF-α), and granulocyte macrophage colony stimulating factor (GM-CSF) by enzyme-linked immunosorbent assay. M-CSF and the soluble cytokine receptors soluble IL-6 receptor (sIL-6r) and soluble TNF receptor type 1 (sTNFr1) were also measured in serum and M-CSF in stimulated WB cultures. Measurements were corrected for mononuclear cell count. We also measured serum bone-specific alkaline phosphatase (ibAP) in all subjects. We found that LPS stimulated secretion of all cytokines both in WB and isolated cell cultures, and that this was attenuated by hydrocortisone. A significantly higher ratio of IL-1β/IL-1ra (p = 0.02) in LPS stimulated WB cultures was seen in the untreated women. Levels of IL-1β and IL-1α measured in WB cultures were lower and IL-1ra was higher in the ERT-treated group but these results were not significant. BAP was higher in the untreated group (p = 0.005) and correlated with IL-α/IL-1ra in the whole group (r = 0.49, p = 0.03). Results of other measurements showed no significant differences between groups. We conclude that estrogen may prevent bone loss following the menopause by altering the balance between IL-1β and IL-1ra.
Heterocycle-Containing Bisphosphonates Cause Apoptosis and Inhibit Bone Resorption by Preventing Protein Prenylation: Evidence from Structure-Activity Relationships in J774 Macrophages
Authors: Luckman Steven P. , Coxon Fraser P. , Ebetino Frank H. , Russell R. Graham G. , Rogers Michael J.
Published: April 2006

Recent evidence suggests that bisphosphonates (BPs) may inhibit bone resorption by mechanisms that lead to osteoclast apoptosis. We have previously shown that BPs also reduce cell viability and induce apoptosis in the macrophage-like cell line J774. To determine whether BPs inhibit osteoclast-mediated bone resorption and affect J774 macrophages by the same molecular mechanism, we examined the potency to reduce J774 cell viability of pairs of nitrogen-containing BPs that differ slightly in the structure of the heterocycle-containing side chain but that differ markedly in antiresorptive potency. In all cases, the most potent antiresorptive BP of each pair also caused the greatest loss of J774 viability, while the less potent antiresorptive BPs were also less potent at reducing J774 cell viability. Similarly, the bisphosphinate, phosphonoalkylphosphinate and monophosphonate analogs of BPs (in which one or both phosphonate groups are modified, giving rise to much less potent or inactive antiresorptive agents) were much less potent or inactive at reducing J774 cell viability. Thus, the structure-activity relationships of BPs for inhibiting bone resorption match those for causing loss of cell viability in J774 cells, indicating that BPs inhibit osteoclast-mediated bone resorption and reduce J774 macrophage viability by the same molecular mechanism. Loss of J774 cell viability after treatment with BPs was associated with a parallel increase in apoptotic cell death. We have recently proposed that nitrogen-containing BPs reduce cell viability and cause J774 apoptosis as a consequence of inhibition of enzymes of the mevalonate pathway and hence loss of prenylated proteins. In this study, the BPs that were potent inducers of J774 apoptosis and potent antiresorptive agents were also found to be effective inhibitors of protein prenylation in J774 macrophages, whereas the less potent BP analogs did not inhibit protein prenylation. This provides strong evidence that BPs with a heterocyclic, nitrogen-containing side chain, such as risedronate, inhibit osteoclast-mediated bone resorption and induce J774 apoptosis by preventing protein prenylation.
Changes in Renal Hemodynamics and Tubular Function of Surgically Cured Primary Hyperparathyroid Patients Are Probably Due to Chronic Hypercalcemic Nephropathy
Authors: Fleiuss De Farias Maria Lucia , Delgado Alvimar G. , Rosenthal Doris , Lazarevitch Maria José , Lima Maurício B. , Vieira José Gilberto H. , Ornellas José Francisco R.
Published: April 2006

To understand the mechanisms responsible for the persistent hypercalciuria and reduced glomerular filtration rate (GFR) previously found in 6 of 10 patients surgically cured of primary hyperparathyroidism (PHPx), the tubular handling of lithium, sodium, calcium, and phosphate as well as the renal hemodynamics were evaluated in these 10 PHPx patients, in 10 control subjects, and in 5 patients with renal hypercalciuria (RH), during fasting and after an oral calcium load. A positive correlation between the fractional excretions of calcium and sodium was found in all groups, but the PHPx patients excreted more calcium for the same amount of sodium than control subjects. The fractional proximal sodium reabsorption (FPRNa), distal delivery, and fractional phosphate reabsorption were similar in all groups; a significant positive correlation was found between the fractional calcium reabsorption and the FPRNa, indicating that proximal tubular function was preserved and that the urinary calcium losses in RH and in the hypercalciuric PHPx patients (h-PHPx) occurred in the distal nephron. However, only h-PHPx patients had reduced renal plasma flow, renal blood flow, and GFR, as well as a high renal vascular resistance, which was even more evident after the calcium challenge. These findings lead us to conclude that RH and h-PHPx patients are very different, as far as kidney dysfunction is concerned, and that a hypercalcemic nephropathy is the most probable cause of the alterations in distal calcium reabsorption and renal hemodynamics found in the h-PHPx patients.
Gap Junctional Intercellular Communication Contributes to the Contraction of Rat Osteoblast Populated Collagen Lattices
Authors: Bowman N. N. , Donahue H. J. , Ehrlich H. P.
Published: April 2006

The contraction of native collagen lattices by resident mesenchymal cells mimics the organization of collagen during development and repair. Lattice contraction is cell density dependent, suggesting that cell-to-cell communications may contribute to the process. This possibility was investigated by comparing lattice contraction by four rat osteoblastic cell lines: ROS 17/2.8 cells (ROS); ROS transfected with an antisense cDNA sequence of the gap junctional protein connexin 43 (RCx16); ROS transfected with connexin 45 cDNA, a connexin not normally expressed in ROS cells (ROS/Cx45); and ROS transfected with cDNA encoding carboxy-terminal truncated Cx45 (ROS/Cx45tr). The cell coupling indices, which reflect gap junctional communication, were quantitated by the fluorescent dye scrape loading. ROS cells were well coupled (index 3.0), ROS/Cx45tr were better coupled (index 4.2), ROS/Cx45 were poorly coupled (index 1.7), and RCx16 showed no coupling (index 1.1). As determined by immunoblotting, the level of connexin 43 protein was increased in both ROS/Cx45tr and ROS/Cx45 cell lines compared with ROS cells, while the level in RCx16 cells was reduced. ROS populated collagen lattices (PCLs) contracted significantly more at day 5 (177 mm2 to 67 mm2) than ROS/Cx45tr (84 mm2), ROS/Cx45 (108 mm2), or RCx16 (114 mm2). Myosin ATPase activity, which is required for lattice contraction, was equivalent in all four cell lines, indicating that it was not responsible for inhibiting PCL contraction. ROS cells in collagen appeared elongated compared with the other cell lines which were more rounded. These experiments suggest gap junctional communication contributes to PCL contraction by resident osteoblasts.
Asymmetric Distribution of Functional Sodium-Calcium Exchanger in Primary Osteoblasts
Authors: Stains Joseph P. , Gay Carol V.
Published: April 2006

To understand calcium translocation in osteoblasts, we have determined the location of sodium-calcium (Na-Ca) exchanger (NCX) in relation to actin and α-tubulin in primary cultures of avian osteoblasts. Osteoblasts derived from the periosteal surface of tibias from growing chickens were cultured for 8 days in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Lysates immunoblotted with antibodies raised against the canine cardiac Na-Ca antibodies revealed a 70 kDa exchanger protein. Cross-reactivity of the anti-NCX antibody was confirmed by enriching for NCX in protein samples derived from plasma membrane vesicles by affinity chromatography using the exchanger inhibitory peptide. Fractions enriched for the exchanger were eluted from the column and subjected to immunoblotting with the anti-NCX antibody, revealing an intense single band at 70 kDa. Examination of live cells loaded with Calcium Green-1 AM ester by confocal microscopy demonstrated sodium-dependent calcium uptake, confirming the presence of functional NCX in intact cells. Immunolocalization studies of osteoblasts stained with anti-NCX antibodies revealed asymmetric localization of the exchanger in cultured osteoblasts, residing almost entirely within two 0.5-μm optical sections along the substrate adherent side of the cells. Since NCX is known to be a low-affinity, high-capacity calcium translocating molecule and also appears to be asymmetrically positioned, it is likely to play a key role in bone formation.
Hypophosphatemic Rickets Accompanying Congenital Microvillous Atrophy
Authors: Kagitani Kuriko , Dr. Yamamoto Takehisa , Miki Kazunori , Matsumoto Sayuri , Shima Masaaki , Tajiri Hitoshi , Harada Tokuzo , Okada Shintaro
Published: April 2006

This report concerns an 11-year-old boy who manifested hypophosphatemic rickets associated with congenital microvillous atrophy (CMA). He had been suffering from vomiting and severe diarrhea from the first day of life and had been treated with total parenteral nutrition (TPN) since he was 67 days old. At 4 years of age, intestinal biopsy resulted in a diagnosis of CMA. He was admitted to our hospital complaining of leg pain at the age of 11. Laboratory data revealed hypophosphatemia, elevated serum 1,25-dihydroxyvitamin D (1,25(OH)2D) levels, and hypercalciuria. A roentgenogram showed rickets in the extremities. A balance study of phosphate in urine and stool indicated that the amount of phosphate leaking into the stool was greater than that into the urine. Moreover, the total amount of phosphate leaking from both the intestine and kidney exceeded the amount of phosphate intake from TPN. The rickets was healed by increasing the phosphate concentration in TPN. This case is different from X-linked hypophosphatemic rickets but similar to hereditary hypophosphatemic rickets with hypercalciuria (HHRH) in terms of hypercalciuria and elevated serum 1,25(OH)2D levels. The effectiveness of phosphate treatments used here is also similar to that used for HHRH. However, this type of hypophosphatemic rickets is unique in that phosphate leaking into the intestine plays an important role in its pathogenesis.
Cartilage-Derived Morphogenetic Proteins and Osteogenic Protein-1 Differentially Regulate Osteogenesis
Authors: Erlacher Ludwig , Mccartney John , Piek Ester , Ten Dijke Peter , Yanagishita Masaki , Oppermann Hermann , Dr. Luyten Frank P.
Published: April 2006

Cartilage-derived morphogenetic proteins-1 and -2 (CDMP-1 and CDMP-2) are members of the bone morphogenetic protein (BMP) family, which play important roles in embryonic skeletal development. We studied the biological activities of recombinant CDMP-1 and CDMP-2 in chondrogenic and osteogenic differentiation and investigated their binding properties to type I and type II serine/threonine kinase receptors. In vivo, CDMP-1 and CDMP-2 were capable of inducing dose-dependently de novo cartilage and bone formation in an ectopic implantation assay. In vitro studies using primary chondrocyte cultures showed that both CDMP-1 and CDMP-2 stimulated equally de novo synthesis of proteoglycan aggrecan in a concentration-dependent manner. This activity was equipotent when compared with osteogenic protein-1 (OP-1). In contrast, CDMPs were less stimulatory than OP-1 in osteogenic differentiation as evaluated by alkaline phosphatase activity and expression levels of bone markers in ATDC5, ROB-C26, and MC3T3-E1 cells. CDMP-2 was the least osteogenic in these assays. Receptor binding studies of CDMP-1 and CDMP-2 revealed that both have affinity for the BMP receptor type IB (BMPR-IB) and BMPR-II, and weakly for BMPR-IA. Moreover, using a promoter/reporter construct, transcriptional activation signal was transduced by BMPR-IB in the presence of BMPR-II upon CDMP-1 and CDMP-2 binding. Our data show that distinct members of the BMP family differentially regulate the progression in the osteogenic lineage, and this may be due to their selective affinity for specific receptor complexes.
Differential Effects of Estrogen Metabolites on Bone and Reproductive Tissues of Ovariectomized Rats
Authors: Westerlind Kim Caroline , Gibson Kristin J. , Malone Patricia , Evans Glenda L. , Turner Russell T.
Published: April 2006

The effects of 17β-estradiol and the important estrogen metabolites, 2-hydroxyestrone (2-OHE1) and 16α-hydroxyestrone (16α-OHE1) on bone, mammary gland, and uterine histology, and on blood cholesterol were investigated in ovariectomized growing rats. Rats were treated with 200 μg/kg of body weight/day of each of the test compounds for 3 weeks. Ovariectomy resulted in uterine and mammary gland atrophy, increased body weight, bone turnover and tibia growth, and hypercholesterolemia. 17β-estradiol treatment prevented these changes, with the exception that this high dose of estrogen did not prevent hypercholesterolemia. 2-OHE1 had no effect on any of the measurements. 16α-OHE1 resulted in bone measurements that did not differ from the 17β-estradiol–treated rats and prevented the increase in serum cholesterol. In contrast, 16α-OHE1 resulted in increases in uterine weight, uterine epithelial cell height, and mammary gland cell proliferation that were significantly less than the 17β-estradiol treatment. These findings demonstrate that 16α-hydroxylation of estrone results in tissue-selective estrogen agonistic activity, whereas 2-hydroxylation resulted in no measured activity. Furthermore, they suggest that factors that modulate the synthesis of these metabolites could selectively influence estrogen target tissues.
Risedronate in the Treatment of Paget's Disease of Bone: An Open Label, Multicenter Study
Authors: Siris Ethel S. , Chines Arkadi A. , Altman Roy D. , Brown Jacques P. , Johnston C Conrad , Lang Robert , Mcclung Michael R. , Mallette Lawrence E. , Miller Paul D. , Ryan Will G. , Singer Frederick R. , Tucci Joseph R. , Eusebio Rachelle A. , Bekker Pirow J.
Published: April 2006

An open-label, multicenter study was conducted to determine the efficacy and safety of oral risedronate (a pyridinyl bisphosphonate) in 162 patients (102 men, 60 postmenopausal women; mean age, 68 years) with moderate to severe Paget's disease of bone (mean serum alkaline phosphatase [ALP] approximately seven times the upper limit of normal). Patients were treated with oral risedronate, 30 mg/day for 84 days, followed by 112 days without treatment. This 196-day cycle was repeated once if serum ALP did not normalize or increased from the nadir value by ≥25%. At the end of the first and second cycles, the mean percentage decreases for serum ALP were 65.7% and 69.1%, and for urinary hydroxyproline/creatinine 50.4% and 66.9%, respectively. The decreases from baseline in ALP and urinary hydroxyproline/creatinine were significant (p < 0.001). Normalization of serum ALP was observed in 86 patients (53.8%): 53 during the first treatment cycle and 33 during the second. There was a significant proportion of patients reporting a decrease in the pagetic bone pain at days 84 and 196 (p < 0.001). Overall, risedronate was well tolerated. Five patients withdrew due to adverse events, none of which were considered to be drug related. In conclusion, 30 mg of oral risedronate administered daily for 84 days significantly reduced the biochemical indices of disease activity and was associated with pain reduction in patients with moderate to severe Paget's disease of bone. Normalization of ALP was observed in the majority of patients. Repeated administration of risedronate was shown to be beneficial. In general, risedronate was well tolerated and demonstrated a good safety profile.
Paget's Disease of Bone: Evidence for a Susceptibility Locus on Chromosome 18q and for Genetic Heterogeneity
Authors: Haslam Sonya I. , Van Hul Wim , Morales-Piga Antonio , Balemans Wendy , San-Millan J. L. , Nakatsuka Kiyoshi , Willems Patrick , Haites Neva E. , Ralston Stuart H.
Published: April 2006

Paget's disease of bone is a common condition characterized by bone pain, deformity, pathological fracture, and an increased incidence of osteosarcoma. Genetic factors play a role in the pathogenesis of Paget's disease but the molecular basis of the disease remains unclear. Previous genetic linkage studies have mapped the rare Paget's disease-like bone dysplasia familial expansile osteolysis (FEO) to chromosome 18q21–22, and recent work has shown evidence of linkage between this locus and Paget's disease in one family. Here we studied the relationship between the 18q21–22 locus and Paget's disease in eight large multiplex families from diverse ethnic backgrounds with inherited Paget's disease. Paget's disease was inherited as an autosomal dominant trait in all families, with high penetrance by the sixth decade. Analysis of seven highly polymorphic markers from chromosome 18q21–22 showed positive summated two-point log10 odds ratio (lodscores) of +2.97 with the marker D18S42 at a recombination fraction (θ) = 0.05, and of +2.95 with the marker D18S60 at θ = 0.00, values which are close to the cut-off of +3.0, which is generally accepted as evidence of linkage. Segregation analysis of the haplotypes and formal statistical analysis using the HOMOG program provided evidence for genetic heterogeneity, however, with evidence for linkage in five families and against linkage in the remaining three families (chi square 8.82; df = 2; p < 0.025). Multipoint linkage analysis in the five linked families showed lodscores of above +3.5 across the whole susceptibility region and a maximum summated lodscore of 3.89 at the marker D18S465. In the three nonlinked families, negative multipoint results were obtained for the whole region, with lodscores below –2.0 in one family, excluding this as a candidate locus for the disease. Our studies demonstrate the importance of hereditary factors in the pathogenesis of Paget's disease and confirm evidence of linkage between Paget's disease and chromosome 18q21–22 in some families. This raises the possibility that Paget's disease and FEO may share a common molecular basis, perhaps due to different mutations in the same gene or family of genes. Data from three families did not support evidence of linkage to 18q21–22 however, indicating that Paget's disease is genetically heterogeneous and suggests the presence of at least one additional locus which remains to be discovered.
Fractal Dimension as a Measure of Altered Trabecular Bone in Experimental Inflammatory Arthritis
Authors: Caldwell Curtis B. , Moran Erica L. , Bogoch Earl R.
Published: April 2006

Our previous studies in experimental inflammatory arthritis (EIA) and in human rheumatoid arthritis demonstrated rapid remodeling with a 5-fold increase in bone resorption and bone formation. Normal condylar trabecular bone is typically anisotropic, with its orientation along lines of stress; rapid remodeling in a pathological state could disturb the usual order of trabeculae. This study assessed change in the structure of trabecular bone of the distal femoral epiphysis after induction of EIA, using a measure of “fractal dimension,” which may be considered a quantitative description of the degree of irregularity of complex surfaces. Data was obtained from specimens in which EIA had been induced in the rabbit knee by 10 injections of carrageenan over 49 days. Photographic enlargements of embedded undecalcified cross-sections of the distal femur were digitized, and software written on a Sun workstation was used to define repeatable regions of interest (ROIs) in the images. The ROIs were subjected to fractal analysis by a power law method. The fractal dimension of the trabecular bone pattern within the ROI was estimated by fitting an equation of the form A(ϵ) = λϵ(2–D) to the data. In this equation, A(ϵ) is the area of the “surface” formed by modeling the ROI data as a three-dimensional structure with the grey-level magnitude providing the third dimension, λ is a scaling constant, ϵ is the size of the measuring “tool” used to measure the area, and D is the fractal dimension. A Mann–Whitney U-test applied to the average of the data from all ROIs showed that the two distributions of fractal dimension were significantly different (p < 0.005). There were only two overlaps between data points for arthritis (with these values higher) and normal groups (n = 11 for each group). Since Howship's lacunae were too small to be resolved in the system utilized, we consider the difference in fractal dimension to be primarily related to trabecular surface orientation, rather than to the increased number of asperities (resorptive foci) occurring due to increased turnover in bone affected by inflammatory arthritis. The results suggest that fractal dimension may be a useful tool for assessing the degree of structural damage to trabeculae in conditions similar to EIA.
Bone Marrow Composition and Bone Microarchitecture and Turnover in Blacks and Whites
Authors: Schnitzler Christine M. , Mesquita Julia
Published: April 2006

We examined the relationship between bone histomorphometric variables versus marrow cellularity, marrow adiposity (among hemopoietic cells), and fatty degeneration (areas of only fat) of bone marrow in iliac crest bone samples from 98 normal black (n = 53) and white (n = 45) males and females. We found blacks to have greater marrow cellularity (p = 0.0001), less marrow adiposity (among hemopoietic cells, p = 0.0001), greater values for bone volume (p = 0.030), trabecular thickness (p = 0.002), and static bone turnover variables (osteoid volume, p = 0.001; osteoid surface, p = 0.001; osteoid thickness, p = 0.001; eroded surface, p = 0.0006) than whites. Marrow cellularity correlated positively with static bone turnover variables osteoid volume (r = 0.257, p = 0.011), osteoid surface (r = 0.265, p = 0.008), osteoid thickness (r = 0.217, p = 0.032), and eroded surface (r = 0.273, p = 0.007) when all 98 cases were analyzed together. These findings suggest that marrow cells may influence bone turnover. The extent of fatty degeneration, but not that of adipose tissue, increased with age in blacks (r = 0.476, p = 0.0003) and whites (r = 0.476, p = 0.001), as did bone loss. There was no racial difference in the extent of fatty degeneration. We conclude that the lesser extent of adiposity in blacks is a racial characteristic that is unaffected by aging, whereas fatty degeneration which may have partly occupied space vacated by bone loss, is an aging phenomenon, unrelated to race. Greater bone turnover in blacks may be expected to lead to more frequent renewal of fatigue-damaged bone, which together with sturdier bone structure may contribute to the lower fragility fracture rates in blacks.
Blood Flow Measurements with [15O]H2O and [18F]Fluoride Ion PET in Porcine Vertebrae
Authors: Piert Morand , Zittel Tilman T. , Machulla Hans-Jurgen , Becker Georg Alexander , Jahn Michael , Maier Gerhard , Bares Roland , Becker Horst Dieter
Published: April 2006

A dual positron emission tomography (PET) tracer study with [18F]fluoride and the freely diffusible tracer [15O]H2O was performed to measure the capillary transport of [18F]fluoride and to evaluate the potential of [18F]fluoride ion PET to quantitate bone blood flow. Under the condition of a high predictable single-pass extraction fraction (EF) for [18F]fluoride, the [18F]fluoride ion influx transport constant (K1F), derived from kinetic [18F]fluoride ion PET measurements, can be used to estimate bone blood flow. Bone blood flow was measured in vertebral bodies by dynamic [15O]H2O PET during continuous ventilation with N2O, O2, and Isoflurane (FiO2 = 0.3) in seven adult mini pigs, followed by dynamic [18F]fluoride ion PET. The mean blood flow measured by [15O]H2O (Flow   H   2 O) was 0.145 ± 0.047 ml·minute–1·ml–1 and the mean K1F was 0.118 ± 0.031 ml·minute–1·ml–1, respectively (mean ± SD). Regional analysis showed excellent agreement between Flow   H   2 O and K1F at low flow and a significant underestimation of flow by K1F relative to Flow   H   2 O in regions of normal and elevated flow. The observed relationship between parameters followed the Renkin-Crone distribution. The permeability-surface product was determined as 0.25 minute−1 for vertebral bodies consisting of a mixture of trabecular and cortical bone. We conclude that [18F]fluoride ion PET can be used to estimate bone blood flow in low and normal flow regions, as long as the flow dependency of the EF is taken into consideration. Above blood flow values of 0.2 to 0.35 ml·minute–1·ml–1, the magnitude of K1F is increasingly independent on blood flow because diffusion limits tracer transport.
Biochemical Markers of Bone Turnover and the Volume and the Density of Bone in Children at Different Stages of Sexual Development
Authors: Mora Stefano , Pitukcheewanont Pisit , Kaufman Francine R. , Nelson Jerald C. , Gilsanz Vicente
Published: April 2006

Bone mass and biochemical markers of bone turnover increase significantly during puberty. We studied the possible relationships between markers of bone formation and bone resorption and increases in skeletal size, bone volume, and bone density in healthy children at different stages of sexual development. Serum concentrations of bone specific alkaline phosphatase (BALP) and osteocalcin (bone Gla protein, BGP), urinary levels of pyridinoline (Pyr) and deoxypyridinoline (Dpyr) and computed tomography (CT) measurements of the cross-sectional areas of the vertebrae and the femurs, the apparent density of cancellous bone in the vertebrae, and the volume and the material density of cortical bone in the femurs were determined in 126 boys and 143 girls, ages 7–18 years. Serum levels of BALP and BGP and urinary concentrations of Pyr and Dpyr peaked in early puberty and were lowest in the later stages of puberty. CT measurements for the cross-sectional areas of the vertebrae and the femurs, the femoral cortical bone areas, and the apparent density of cancellous bone increased in all children during puberty, while values for material bone density did not change significantly with the stage of sexual development. BALP and BGP showed significant inverse correlations with the material density of bone (r = –0.23 and –0.24, respectively), but no association with bone volume in the appendicular or axial skeleton. In contrast, Pyr and Dpyr correlated with femoral cross-sectional area (r = –0.24 and –0.33, respectively) and cortical bone area (r = –0.29 and –0.33, respectively), and with the apparent density of vertebral cancellous bone (r = –0.26 and –0.19, respectively), but not with the material density of bone. We conclude that, during puberty, there is a differential association between the two components of bone mass and the markers of bone formation and bone resorption; while markers of bone formation are related to the material density of bone, markers of bone resorption are related to the volume of bone.
A Six-Year Longitudinal Study of the Relationship of Physical Activity to Bone Mineral Accrual in Growing Children: The University of Saskatchewan Bone Mineral Accrual Study
Authors: Bailey D. A. , Mckay H. A. , Mirwald R. L. , Crocker P. R. E. , Faulkner R. A.
Published: April 2006

To investigate the influence of physical activity on bone mineral accrual during the adolescent years, we analyzed 6 years of data from 53 girls and 60 boys. Physical activity, dietary intakes, and anthropometry were measured every 6 months and dual-energy X-ray absorptiometry scans of the total body (TB), lumbar spine (LS), and proximal femur (Hologic 2000, array mode) were collected annually. Distance and velocity curves for height and bone mineral content (BMC) were fitted for each child at several skeletal sites using a cubic spline procedure, from which ages at peak height velocity (PHV) and peak BMC velocity (PBMCV) were identified. A mean age- and gender-specific standardized activity (Z) score was calculated for each subject based on multiple yearly activity assessments collected up until age of PHV. This score was used to identify active (top quartile), average (middle 2 quartiles), or inactive (bottom quartile) groups. Two-way analysis of covariance, with height and weight at PHV controlled for, demonstrated significant physical activity and gender main effects (but no interaction) for PBMCV, for BMC accrued for 2 years around peak velocity, and for BMC at 1 year post-PBMCV for the TB and femoral neck and for physical activity but not gender at the LS (all p < 0.05). Controlling for maturational and size differences between groups, we noted a 9% and 17% greater TB BMC for active boys and girls, respectively, over their inactive peers 1 year after the age of PBMCV. We also estimated that, on average, 26% of adult TB bone mineral was accrued during the 2 years around PBMCV.
Femoral Neck Is a Sensitive Indicator of Bone Loss in Immobilized Hind Limb of Mouse
Authors: Jämsä Timo , Koivukangas Antti , Ryhänen Jorma , Jalovaara Pekka , Tuukkanen Juha
Published: April 2006

The present study was carried out to evaluate a unilateral hind limb immobilization model in the mouse. The right legs of male mice (age 10–12 weeks) were immobilized for 3 weeks against the abdomen by an elastic bandage. Body weight decreased significantly during the immobilization. Peripheral quantitative computed tomography (pQCT) analysis showed that the cross-sectional cortical area (CSA), the bone mineral content (BMC), and the bone mineral density (BMD) of the tibial diaphysis were lower in both legs of the immobilized animals than in age-matched controls, but the difference was mainly due to weight reduction. At the tibial metaphysis, CSA, BMC, and BMD were reduced in both legs of the immobilized animals, even after weight adjustment. At the femoral neck, CSA, BMC, and BMD were significantly lower in both legs of the immobilized animals, and the difference between the hind legs of the immobilized animals was also highly significant. The findings of the pQCT study were in good agreement with the changes in mechanical strength. The tibia was a more sensitive indicator of diaphyseal bone weakening than the femur when measuring the bending breaking force of the diaphysis. The femoral neck showed significantly decreased strength, and the difference between the immobilized leg and the contralateral leg was most clearly seen in lateral loading. We conclude that 3 weeks of hind limb immobilization weakened the tibia and femur significantly compared with their contralateral counterparts. The reduction was more significantly seen in the mechanical bending strength than in the pQCT evaluation, and the femoral neck was the most sensitive indicator of bone weakening.
TNF-α and IL-1β Suppress N-Cadherin Expression in MC3T3-E1 Cells
Authors: Tsutsumimoto Takahiro , Kawasaki Satoshi , Ebara Sohei , Takaoka Kunio
Published: April 2006

Excessive production of tumor necrosis factor (TNF) and interleukin-1 (IL-1) secondary to estrogen deficiency have been implicated as the cause of osteoporosis in postmenopausal woman. These cytokines appear to stimulate osteoclast precursor proliferation and activate mature osteoclast formation directly and possibly indirectly via osteoblasts. To investigate the other possible roles that these cytokines may play in stimulating the bone resorption process, we examined the effect of TNF-α and IL-1β on cell–cell adhesion molecules, cadherins, in osteoblastic MC3T3-E1 cells. In this study, we investigated cadherin expression and the effect of TNF-α, IL-1β, and parathyroid hormone (PTH) on the expression of cadherins in MC3T3-E1 cells. Confluent cultures of MC3T3-E1 cells were challenged with recombinant human TNF-α (1–100 U/ml), recombinant human IL-1β (1–100 ng/ml) and human PTH(1–34) (1–100 ng/ml), respectively. The results show that MC3T3-E1 cells express functional cadherin molecules, N-cadherin and OB-cadherin. TNF-α (10–100 U/ml) and IL-1β (10–100 ng/ml) suppressed N-cadherin without changing OB-cadherin expression, while PTH (1–100 ng/ml) had no effect on cadherin expression. These results raise the possibility that TNF-α and IL-1β may compromise the cell–cell adhesion of osteoblasts which cover the bone surface. The ensuing compromised cell–cell adhesion of osteoblasts may in turn facilitate the direct adhesion of osteoclasts on the calcified bone matrix surface. These results implicate an indirect role for osteoblasts in the promotion of bone resorption by TNF-α and IL-1β.
Comparison of Recombinant Human PTH(1–34) (LY333334) with a C-Terminally Substituted Analog of Human PTH-Related Protein(1–34) (RS-66271): In Vitro Activity and In Vivo Pharmacological Effects in Rats
Authors: Dr. Frolik Charles A. , Cain Ricky L. , Sato Masahiko , Harvey Anita K. , Chandrasekhar S. , Black Elwood C. , Tashjian Armen H. , Hock Janet M.
Published: April 2006

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are believed to exert their biological actions through binding and activation of a common cell surface receptor. Recently, an analog of PTHrP (RS-66271), was described that demonstrated reduced binding affinity for the PTH/PTHrP receptor compared with bovine PTH(1–34) but retained equal biological activity. The present study investigated the receptor binding affinities of synthetic RS-66271 and recombinant human PTH(1–34) (LY333334) and compared their in vitro and in vivo pharmacological effects. RS-66271 had one hundredth the activity of PTH(1–34) in competing for the binding of [125I] [Nle8,18, Tyr34]human PTH(1–34) to the human PTH/PTHrP receptor stably expressed in a human kidney cell line. Despite this reduced binding affinity, RS-66271 had equivalent activity in increasing both cAMP production in osteoblast-like cells and bone resorption in neonatal mouse calvariae. However, RS-66271 was 7.6-fold less active in stimulating inositol phosphate production. For in vivo studies, young, male Fisher rats received a daily subcutaneous dose of either 10 or 40 μg/kg of peptide for 1, 2, or 4 weeks. Volumetric bone mineral density and total bone mineral content of the proximal tibia were determined by peripheral quantitative computerized tomography. Trabecular and cortical bone of the distal femur were analyzed for calcium and dry weight. Lumbar vertebrae (L4–L6) were analyzed by histomorphometry. Trabecular and cortical bone mass showed a dose- and time-dependent increase in the treated animals compared with the controls. These increases were evident as early as 1 week after initiation of dosing. There were no consistent significant differences in the comparative effects of PTH(1–34) and RS-66271 on the measured bone parameters. In conclusion, despite the reduced binding affinity of RS-66271 for the PTH/PTHrP receptor compared with human PTH(1–34), both peptides displayed similar in vitro and in vivo pharmacological effects.