bluuebunny/crossref_metadata_2025 huggingface dataset:
The Human Vitamin D Receptor Gene (VDR) Is Localized to Region 12cen-q12 by Fluorescent In Situ Hybridization and Radiation Hybrid Mapping: Genetic and Physical VDR Map
Authors: Taymans Susan E. , Pack Svetlana , Pak Evgenia , Orban Zsolt , Barsony Julia , Zhuang Zhengping , Stratakis Constantine A.
Published: April 2006

The vitamin D receptor (VDR) is a member of the steroid hormone receptor superfamily of ligand-activated transcription factors. The VDR gene was previously mapped to human chromosome 12q13–12q14, but its precise physical and genetic localization are unknown. The present study reports the mapping of the human VDR gene by radiation hybrid (RH) analysis, the isolation of a bacterial artificial chromosome (BAC) containing this gene, and physical mapping of the VDR gene by fluorescent in situ hybridization (FISH). RH analysis placed the VDR gene locus at chromosome 12cen-q12, flanked by Stanford Human Genome Center (SHGC) 30216 and SHGC 9798 (D12S1892) markers. FISH analysis of a BAC containing the VDR gene confirmed its centromeric location. Thus, we have identified a BAC and genetic markers which can be used in the genetic analysis of the VDR gene and investigation of its involvement in osteoporosis and related disorders. We conclude that the VDR gene is centromeric to its previously reported locus on chromosome 12.
Direct Three-Dimensional Morphometric Analysis of Human Cancellous Bone: Microstructural Data from Spine, Femur, Iliac Crest, and Calcaneus
Authors: Hildebrand Tor , Laib Andres , Müller Ralph , Dequeker Jan , Rüegsegger Peter
Published: April 2006

The appearance of cancellous bone architecture is different for various skeletal sites and various disease states. During aging and disease, plates are perforated and connecting rods are dissolved. There is a continuous shift from one structural type to the other. So traditional histomorphometric procedures, which are based on a fixed model type, will lead to questionable results. The introduction of three-dimensional (3D) measuring techniques in bone research makes it possible to capture the actual architecture of cancellous bone without assumptions of the structure type. This requires, however, new methods that make direct use of the 3D information. Within the framework of a BIOMED I project of the European Union, we analyzed a total of 260 human bone biopsies taken from five different skeletal sites (femoral head, vertebral bodies L2 and L4, iliac crest, and calcaneus) from 52 donors. The samples were measured three-dimensionally with a microcomputed tomography scanner and subsequently evaluated with both traditional indirect histomorphometric methods and newly developed direct ones. The results show significant differences between the methods and in their relation to the bone volume fraction. Based on the direct 3D analysis of human bone biopsies, it appears that samples with a lower bone mass are primarily characterized by a smaller plate-to-rod ratio, and to a lesser extent by thinner trabecular elements.
Lactulose Stimulates Calcium Absorption in Postmenopausal Women
Authors: van den heuvel Ellen G. H. M. , Muijs Theo , van dokkum Wim , Schaafsma Gertjan
Published: April 2006

Animal studies have indicated that calcium absorption is increased by lactulose, a synthetic disaccharide. Therefore, the influence of lactulose on calcium absorption was measured in postmenopausal women who may benefit from the possible enhancing effect of lactulose on calcium absorption. Twelve postmenopausal women drank 100 ml of water containing 5 or 10 g of lactulose or a reference substance at breakfast for 9 days. The three treatments were given according to a randomized, double-blind, cross-over design, separated by two 19-day wash-out periods. On the 8th day of each treatment period,44Ca dissolved in orange juice was drunk immediately after the solution with the study substance and just before a standard breakfast with 162 mg of carrier calcium. Within half an hour,48Ca was given intravenously. Based on isotope ratios measured in urine collected before and until 36 h after isotope administration, true fractional calcium absorption was calculated. Calcium absorption during the treatments with the reference substance, 5 g and 10 g of lactulose was (mean ± SD) 27.7 ± 7.7, 30.0 ± 7.6, and 32.2 ± 7.0, respectively. A significant difference in calcium absorption was found between the highest dose of lactulose and the reference treatment (p < 0.01). A significant linear trend was found between the dose of lactulose and its positive effect on calcium absorption. In conclusion, in postmenopausal women a 9-day consumption of lactulose increases calcium absorption in a dose-response way. More research is warranted to explore how lactulose stimulates calcium absorption and whether it is able to improve calcium balance and/or to attenuate the rate of aging bone loss.
Effects of High-Impact Exercise on Ultrasonic and Biochemical Indices of Skeletal Status: A Prospective Study in Young Male Gymnasts
Authors: Daly Robin M. , Rich Peter A. , Klein Rudi , Bass Shona
Published: April 2006

Physical activity has been proposed as one strategy to enhance bone mineral acquisition during growth. The aim of this study was to determine whether frequent impact loading associated with gymnastics training confers a skeletal benefit on pre- and peripubertal male gymnasts. We measured broadband ultrasonic attenuation (BUA, dB/MHz) at the calcaneus (CBUA); ultrasound velocity (m/s) at the calcaneus (CVOS), distal radius (RVOS) and phalanx (PVOS); serum osteocalcin (OC); total alkaline phosphatase (ALP) and insulin-like growth factor-I (IGF-I) every 3–4 months over an 18-month period in elite male gymnasts and matched normoactive controls (pubertal stage ≤2). Ground reaction forces of common gymnastics maneuvers were determined using a force platform and loading histories of the upper and lower extremities approximated from video recordings. Ultrasound results were expressed as a standardized score (Z score) adjusted for age, height, and weight. At baseline, no differences were detected between the gymnasts (n = 31) and controls (n = 50) for CBUA, although ultrasound velocity at each site was higher in the gymnasts (0.6–1.5 SD) than the predicted mean in controls (p ≤ 0.001). Over 18 months, CBUA Z scores increased significantly in the gymnasts from baseline (0.3 vs. 1.0, p < 0.05, n = 18). In contrast, ultrasound velocity did not increase in either group, although CVOS and RVOS remained significantly higher in gymnasts compared with controls (range p < 0.01 and < 0.001). No differences between groups were found for OC, ALP, or IGF-I at any time. Gymnastics training was associated with on average 102 and 217 impacts per session on the upper and lower extremities, respectively, with peak magnitudes of 3.6 and 10.4 times body weight. These results suggest that frequent high-impact, weight-bearing exercise during the pre and peripubertal period may enhance the mechanical competence of the skeleton, perhaps offering an important strategy for osteoporosis prevention if the benefits are maintained.
Long-Term Recreational Gymnastics, Estrogen Use, and Selected Risk Factors for Osteoporotic Fractures
Authors: Uusi-Rasi Kirsti , Sievänen Harri , Vuori Ilkka , Heinonen Ari , Kannus Pekka , Pasanen Matti , Rinne Marjo , Oja Pekka
Published: April 2006

The purpose of this cross-sectional study was to examine whether long-term participation in recreational gymnastics or folk dancing or estrogen replacement therapy (ERT) is associated with mechanically more competent bones and improved muscular strength and body balance. One hundred and seventeen healthy, female postmenopausal recreational gymnasts (mean age 62.1 [SD 4.7] years) and 116 sedentary controls (mean age 61.5 [4.6] years) were enrolled in the study. Bone mineral content (BMC) of the distal radius, femoral neck, and trochanter were measured with dual-energy X-ray absorptiometry. BMC of the midshaft and distal tibia and trabecular density (TrD) of the distal tibia were measured with peripheral computed quantitative tomography. Maximal isometric strength, muscular power, cardiorespiratory fitness, and body balance of the participants were also assessed. The cardiorespiratory fitness, muscular strength, and dynamic balance of the recreational gymnasts and folk dancers combined were significantly better than those of the controls, the average group difference ranging from 7.5% (95% confidence interval 5.0–9.9%) in dynamic balance to 12.8% (6.6–19.4%) in dynamic muscular power. ERT was not associated with the fitness indicators, muscular power, or balance, but was significantly associated with the BMC at all the measured bone sites, the mean group difference between estrogen users and nonusers ranging from 6.5% (3.7–9.3%) for the tibial shaft to 11.8% (6.4–17.0%) for the distal radius. Recreational gymnastics, in turn, was significantly associated with higher BMC at the tibia only, the mean group difference being 3.9% (0.9–6.9%) for the tibial shaft and 7.7% (3.7–11.9%) for the distal tibia. Recreational gymnastics was also associated with higher TrD at the distal tibia (5.2%; 1.2–9.2%), whereas estrogen usage did not show such association. The results indicate that ERT seems especially effective in preventing postmenopausal bone loss, whereas recreational gymnastics and folk dancing improve muscular performance and body balance in addition to increased bone mass and bone size in the tibia. All these factors are essential in prevention of fall-related fractures of the elderly.
A Chimeric Form of Osteoprotegerin Inhibits Hypercalcemia and Bone Resorption Induced by IL-1β, TNF-α, PTH, PTHrP, and 1,25(OH)2D3
Authors: Morony Sean , Capparelli Casey , Lee Richard , Shimamoto Grant , Boone Thomas , Lacey David L. , Dunstan Colin R.
Published: April 2006

Osteoprotegerin (OPG) is a secreted protein that inhibits osteoclast formation and activity and appears to be a critical regulator of bone mass and metabolism. In the current study, mice were challenged with various cytokines and hormones (interleukin-1β, tumor necrosis factor-α, parathyroid hormone, parathyroid hormone-related protein, and 1α,25-dihydroxyvitamin D3) that are known to increase bone resorption and cause hypercalcemia and treated concurrently with either a recombinant chimeric Fc fusion form of human OPG, with enhanced biological activity (cOPG) (2.5 mg/kg/day) or vehicle. Mice receiving these bone-resorbing factors became hypercalcemic by day 3 after commencing treatment and had increased bone resorption as evidenced by elevated osteoclast numbers on day 5. Concurrent cOPG treatment prevented hypercalcemia (p < 0.05) and maintained osteoclast numbers in the normal range (p < 0.001). The demonstration that cOPG can inhibit bone resorption suggests that this molecule may be useful in the treatment of diseases including hyperparathyroidism, humoral hypercalcemia of malignancy, osteoporosis, and inflammatory bone disease, which are characterized, in part, by increases in osteoclastic bone resorption.
Expression of Osteoprotegerin (Osteoclastogenesis Inhibitory Factor) in Cultures of Human Dental Mesenchymal Cells and Epithelial Cells
Authors: Sakata Masatoshi , Shiba Hideki , Komatsuzawa Hitoshi , Fujita Tsuyoshi , Ohta Kouji , Sugai Motoyuki , Suginaka Hidekazu , Kurihara Hidemi
Published: April 2006

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) inhibits osteoclast differentiation, activity, and survival; therefore OPG/OCIF may regulate the resorption of dental hard tissues, such as alveolar bone, cementum, and dentin. To investigate this issue, reverse transcriptase-polymerase chain reaction using specific primers for OPG/OCIF was performed with total RNAs isolated from human gingival keratinocytes (HGKs), human gingival fibroblasts (HGFs), human periodontal ligament cells (HPDLs), and human pulp cells (HPCs) in culture. PCR products were found in HGFs, HPDLs, and HPCs, but not in HGKs, and the DNA sequence of these products was 100% identical to the reported sequence of the OPG gene. Northern blot analyses also showed that HGFs, HPDLs, and HPCs, but not HGKs, expressed OPG/OCIF transcripts of ∼2.5 kb. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) increased OPG/OCIF mRNA levels in a dose-and time-dependent manner in HPDL. After 12 h of treatment, IL-1β at 3 ng/ml and TNF-α at 3 ng/ml increased OPG/OCIF mRNA expression by 190% and 110%, respectively, with a maximal effect. The stimulatory effects of IL-1β and TNF-α were also seen in HPC. However, IL-6 and transforming growth factor-β had little effect on OPG/OCIF mRNA levels in HPDL. These findings suggest that OPG/OCIF synthesized by dental mesenchymal cells locally regulates the resorption of dental hard tissues through cytokines.
Anabolic or Catabolic Responses of MC3T3-E1 Osteoblastic Cells to Parathyroid Hormone Depend on Time and Duration of Treatment
Authors: Schiller Paul C. , D'Ippolito Gianluca , Roos Bernard A. , Howard Guy A.
Published: April 2006

We have investigated signaling (cAMP) and anabolic responses (mineralization of extracellular matrix [ECM]) to parathyroid hormone (PTH) in long-term (30 days) cultures of MC3T3-E1 cells, a murine model of osteoblast differentiation. Expression of PTH/PTH–related peptide receptor (PTH1R) mRNA is detected early and remains relatively constant for 2 weeks with somewhat higher levels observed during the second half of the culture period. In contrast to the relatively stable PTH1R mRNA expression, the cAMP response to PTH varies markedly with no response at day 5 and a marked response (80-fold versus control) by day 10. Responsiveness to PTH remains elevated with fluctuations of 30- to 80-fold stimulation throughout the remainder of the culture period. The timing and duration of PTH treatment to achieve in vitro mineralization of ECM was evaluated. When continuous PTH treatment was initiated before day 20, mineralization decreased. If continuous PTH treatment began on or after day 20, mineralization was unaffected. However, if treatment began on day 20 and then stopped on day 25, mineralization on day 30 was increased 5-fold. This mineralization response to intermittent PTH was confirmed in primary cultures of murine and human osteoblastic cells. These data provide a potential basis for understanding the differential responses to PTH (anabolic versus catabolic) and indicate the developmental temporal variance of anabolic and catabolic responses. Since cAMP signaling was relatively unchanged during this interval (day 10–30) and stimulation of adenylate cyclase only partially mimicked the PTH effect on increased mineralization, other signaling pathways are likely to be involved in order to determine the specific anabolic response to short-term PTH treatment during the differentiation process.
Low Cell Motility Induced by hsp27 Overexpression Decreases Osteolytic Bone Metastases of Human Breast Cancer Cells In Vivo
Authors: Lemieux Pierre , Harvey J. , Guise Theresa , Dallas Mark , Oesterreich Steffi , Yin Juan Juan , Selander Katri , Fuqua Suzanne
Published: April 2006

The mechanisms controlling the formation of osteolytic bone metastases in patients with breast cancer are still poorly understood. To explore the role of motility in the establishment of osteolytic bone metastases, we have used a model of bone metastasis in which MDA-MB-231 breast cancer cells exhibiting low (hsp27-transfectants) and high (control-transfectant) endogenous cell motility were compared. We found that MDA-MB-231 cells exhibiting low cell motility were less capable of establishing osteolytic lesions. The number and the area of the osteolytic lesions in mice inoculated with low motility cells were both significantly smaller. Histomorphometry of bone lesions also demonstrated less tumor area in mice bearing hsp27 transfectants although there was no difference in the osteoclast number per square millimeter of tumor–bone interface. These data suggest that cell motility may be an important mechanism in the metastatic cascade of breast cancer cells to the bone and that controlling cell motility may be a useful target to prevent the establishment of osteolytic bone metastases.
Trabecular Bone Turnover and Bone Marrow Cell Development in Tail-Suspended Mice
Authors: Sakata Takeshi , Sakai Akinori , Tsurukami Hiroshi , Okimoto Nobukazu , Okazaki Yuichi , Ikeda Satoshi , Norimura Toshiyuki , Nakamura Toshitaka
Published: April 2006

To clarify the relationship between the changes of trabecular bone turnover and bone marrow cell development during mechanical unloading and reloading, we performed experiments with tail-suspended mice. At 8 weeks of age, 150 male ddY mice were divided into three body weight-matched groups. Mice of group 1 were euthanized at the start of tail suspension (day 0) as a baseline control. The mice of group 2 were subjected to hindlimb unloading by tail suspension for 14 days and reloading for the subsequent 14 days. The mice of group 3 were normally loaded as age-matched controls. Mice of groups 2 and 3 were sacrificed at 7, 14, and 28 days after the start of the experiment. In the first experiment (histomorphometric study of tibiae), unloading for 7 and 14 days and reloading for the subsequent 14 days significantly decreased the bone volume compared with that in the age-matched controls, respectively. Unloading for 7 and 14 days also significantly reduced the bone formation rate (BFR/BS), respectively, but reloading for the subsequent 14 days restored BFR/BS to the control level. While the unloading for 7 and 14 days significantly increased both the osteoclast surface (Oc.S/BS) and the osteoclast number (Oc.N/BS), the reloading for the subsequent 14 days decreased Oc.S/BS and Oc.N/BS, respectively. In the second experiment (bone marrow cell culture study of tibiae), unloading for 7 and 14 days reduced the adherent stromal cell number, without significance. Unloading for 7 days significantly decreased the mineralized nodule formation. Reloading for the subsequent 14 days markedly increased the adherent stromal cell number and the mineralized nodule formation. Unloading for 7 days significantly increased the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. These data clearly demonstrate that unloading reduces bone formation and increases bone resorption, and subsequent reloading restores reduced bone formation and suppresses increased bone resorption, closely associated with the changes in adherent stromal cell number, mineralized nodule formation, and the number of TRAP-positive multinucleated cells.
Low Body Mass Index Is an Important Risk Factor for Low Bone Mass and Increased Bone Loss in Early Postmenopausal Women
Authors: Ravn P. , Cizza G. , Bjarnason N. H. , Thompson D. , Daley M. , Wasnich R. D. , Mcclung M. , Hosking D. , Yates A. J. , Christiansen C.
Published: April 2006

Thinness (low percentage of body fat, low body mass index [BMI], or low body weight) was evaluated as a risk factor for low bone mineral density (BMD) or increased bone loss in a randomized trial of alendronate for prevention of osteoporosis in recently postmenopausal women with normal bone mass (n = 1609). The 2-year data from the placebo group were used (n = 417). Percentage of body fat, BMI, and body weight were correlated with baseline BMD (r = −0.13 to −0.43, p < 0.01) and 2-year bone loss (r = −0.14 to −0.19, p < 0.01). Women in the lowest tertiles of percentage of body fat or BMI had up to 12% lower BMD at baseline and a more than 2-fold higher 2-year bone loss as compared with women in the highest tertiles (p ≤ 0.004). Women with a lower percentage of body fat or BMI had higher baseline levels of urine N-telopeptide cross-links (r = −0.24 to −0.31, p < 0.0001) and serum osteocalcin (r = −0.12 to −0.15, p < 0.01). To determine if the magnitude of treatment effect of alendronate was dependent on these risk factors, the group treated with 5 mg of alendronate was included (n = 403). There were no associations between fat mass parameters and response to alendronate treatment, which indicated that risk of low bone mass and increased bone loss caused by thinness could be compensated by alendronate treatment. In conclusion, thinness is an important risk factor for low bone mass and increased bone loss in postmenopausal women. Because the response to alendronate treatment is independent of fat mass parameters, prevention of postmenopausal osteoporosis can be equally achieved in thinner and heavier women.
The Effect of Pregnancy on Bone Density and Bone Turnover
Authors: Naylor K. E. , Iqbal P. , Fledelius C. , Fraser R. B. , Eastell R.
Published: April 2006

During pregnancy, the mother adapts to meet the calcium demands of the fetus. The effect of this adaptation on the maternal skeleton is not fully understood. Our objectives were to evaluate changes in bone mineral density (BMD) and bone turnover during pregnancy. We studied 16 women longitudinally, with baseline measurements before pregnancy; then at 16, 26, and 36 weeks of pregnancy; and postpartum. We measured total-body BMD and biochemical markers of bone resorption (urinary pyridinium crosslinks and telopeptides of type I collagen) and bone formation (serum bone alkaline phosphatase, propeptides of type I procollagen [PINP] and osteocalcin). We also measured parathyroid hormone (PTH), insulin-like growth factor I (IGF-I), and human placental lactogen. Postpartum, BMD increased in the arms (2.8%, P< 0.01) and legs (1.9%, P< 0.01) but decreased in the pelvis (−3.2%, P<0.05) and spine (−4.6%, P< 0.01) compared with prepregnancy values. All biochemical markers, with the exception of osteocalcin concentration, increased during pregnancy. The change in IGF-I at 36 weeks was related to the change in biochemical markers (e.g., PINP, r = 0.72, P= 0.002). Pregnancy is a high-bone-turnover state. IGF-I levels may be an important determinant of bone turnover during pregnancy. Elevated bone turnover may explain trabecular bone loss during pregnancy.
1,25-Dihydroxyvitamin D3 as Well as Its Analogue OCT Lower Blood Calcium Through Inhibition of Bone Resorption in Hypercalcemic Rats with Continuous Parathyroid Hormone–Related Peptide Infusion
Authors: Endo Ko-Ichi , Katsumata Kyoko , Hirata Michinori , Masaki Toshimi , Kubodera Noboru , Nakamura Toshitaka , Ikeda Kyoji , Ogata Etsuro
Published: April 2006

The effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and its analogue 22-oxa-1,25(OH)2D3 (22-oxacalcitriol) (OCT) on calcium and bone metabolism were examined in an animal model of hypercalcemia with continuous infusion of parathyroid hormone–related peptide (PTHrP), to determine whether active vitamin D could counteract the skeletal action of PTHrP in addition to its reported effect in suppressing the production of PTHrP in cancer cells. Parathyroid glands were removed from 8-week-old Sprague–Dawley rats to eliminate the confounding effects of endogenous PTH. Animals were then continuously infused with human PTHrP(1–34) at a constant rate via osmotic minipumps for 2 weeks, and at the same time treated orally or intravenously with OCT or 1,25(OH)2D3 four to nine times during the 2-week period. Under these conditions, OCT and, surprisingly, 1,25(OH)2D3 alleviated hypercalcemia in a dose-dependent manner. 1,25(OH)2D3 and OCT suppressed the urinary excretion of deoxypyridinoline, although they did not affect renal calcium handling, suggesting that the antihypercalcemic effect is attributable to the inhibition of bone resorption. These active vitamin D compounds also counteracted the effects of PTHrP at the proximal renal tubules, as reflected by a decrease in phosphate excretion. Histomorphometric analysis of bone revealed a dose-related decrease in parameters of bone resorption. These results suggest that 1,25(OH)2D3 as well as OCT has the potential to alleviate hypercalcemia, at least in part, through the inhibition of bone resorption in hypercalcemic rats with constant PTHrP levels. We propose that the main function of active vitamin D in high bone-turnover states is to inhibit bone resorption, and this may have important implications for the understanding of the role of active vitamin D in the treatment of metabolic bone diseases, such as osteoporosis.
Cementum-Forming Cells Are Phenotypically Distinct from Bone-Forming Cells
Authors: Grzesik Wojciech J. , Cheng Hui , Oh Julia S. , Kuznetsov Sergei A. , Mankani Mahesh H. , Uzawa Katsuhiro , Robey Pamela Gehron , Yamauchi Mitsuo
Published: April 2006

Normal human cementum-derived cells (HCDCs), expanded in vitro, formed mineralized matrix when attached to a ceramic carrier and transplanted subcutaneously into immunodeficient mice. The mineralized matrix elaborated by transplanted HCDC exhibited several features identical to cementum in situ and was significantly different from bone deposited by similarly transplanted human bone marrow stromal cells (BMSCs). No bone marrow formation and very few or no tartrate-resistant acid phosphatase (TRAP)-positive cells (osteoclasts and osteoclastic precursors) were found in HCDC transplants. In contrast, in BMSC transplants both hematopoiesis and TRAP-positive cells were routinely observed. Furthermore, compared with BMSC-derived matrix, HCDC-derived matrix was less cellular, numerous empty lacunae were present, and fewer cells were found on the cementum matrix/ceramic carrier interface. The organization of collagen fibers in HCDC-derived matrix, as visualized by using the Picrosirus red staining method, was similar to cementum, with typical unorganized bundles of collagen fibers. In contrast, bone matrix elaborated by transplanted BMSC had lamellar structure, identical to mature bone in situ. Finally, cementocytes embedded in the cementum-like matrix were immunopositive for fibromodulin and lumican, whereas osteocytes within the bonelike matrix were negative. This pattern is consistent with the cementum and bone in situ, respectively. These results indicate that human cementum cells are phenotypically distinct from bone cells and provide further validation of the combined in vitro/in vivo model of human cementogenesis recently developed in our laboratory.
Stimulation of Sodium-Dependent Phosphate Transport and Signaling Mechanisms Induced by Basic Fibroblast Growth Factor in MC3T3-E1 Osteoblast-like Cells
Authors: Suzuki Atsushi , Palmer Gaby , Bonjour Jean-Philippe , Caverzasio Joseph
Published: April 2006

Physiological and pathological observations indicate that basic fibroblast growth factor (bFGF) is an important regulator of osteoblastic cell differentiation and in particular of cranial ossification. Experimental evidence suggests that inorganic phosphate (Pi) transport could be an important function of bone matrix calcification. In the present study, we address the influence of bFGF on Pi transport activity in MC3T3-E1 osteoblast-like cells derived from mouse calvaria. The results indicate that bFGF is a potent and selective stimulator of sodium-dependent Pi transport in these cells. The change in Pi transport activity induced by bFGF depends on transcription and translation and corresponds to a change in the maximum velocity of the Pi transport system (Vmax). These observations suggest that enhanced Pi transport activity in response to bFGF may result from insertion of newly synthesized Pi transporters into the plasma membrane. A selective inhibitor of fibroblast growth factor receptor (FGFR) tyrosine kinase, SU5402, blunted the stimulation of Pi transport induced by bFGF. It also prevented the increase in protein tyrosine phosphorylation induced by bFGF, including phosphorylation of FGFR-1, FGFR-2, phospholipase C-γ (PLC-γ), and Shc as well as the recruitment of the Grb2/Sos signaling complex. In addition, bFGF- induced the activation of the mitogen-activated protein (MAP) kinases extracellular signal–regulated kinase (ERK) and p38, effects that were prevented by SU5402. Both the protein kinase C (PKC) inhibitor calphostin C and PKC down-regulation suppressed the stimulatory effect of bFGF on Pi transport. Selective inhibitors of ERK and p38 MAP kinases slightly reduced this cellular response with a significant effect observed with the highest concentration of the p38 MAP kinase inhibitor. In conclusion, the results of this study indicate that bFGF selectively stimulates Pi transport in calvaria-derived osteoblastic cells. The main signaling mechanism responsible for this effect involves tyrosine phosphorylation of PLC-γ and activation of PKC, with a possible contribution of the p38 MAP kinase pathway.
Heterogeneity in the Growth of the Axial and Appendicular Skeleton in Boys: Implications for the Pathogenesis of Bone Fragility in Men
Authors: Bradney Michelle , Karlsson Magnus K. , Duan Yunbo , Stuckey Stephen , Bass Shona , Seeman Ego
Published: April 2006

Men with spine fractures have reduced vertebral body (VB) volume and volumetric bone mineral density (vBMD). Men with hip fractures have reduced femoral neck (FN) volume and vBMD, site-specific deficits that may have their origins in growth. To describe the tempo of growth in regional bone size, bone mineral content (BMC), and vBMD, we measured bone length, periosteal and endocortical diameters, BMC, and vBMD using dual-energy X-ray absorptiometry in 184 boys aged between 7 and 17 years. Before puberty, growth was more rapid in the legs than in the trunk. During puberty, leg growth slowed while trunk length accelerated. Bone size was more advanced than BMC in all regions, being ∼70% and ∼35% of their predicted peaks at 7 years of age, respectively. At 16 years of age, bone size had reached its adult peak while BMC was still 10% below its predicted peak. The legs accounted for 48%, whereas the spine accounted for 10%, of the 1878 g BMC accrued between 7 and 17 years. Peripubertal growth contributed (i) 55% of the increase in leg length but 78% of the mineral accrued and (ii) 69% of the increase in spine length but 87% of the mineral accrued. Increased metacarpal and midfemoral cortical thickness was caused by respective periosteal expansion with minimal change in the endocortical diameter. Total femur and VB vBMD increased by 30–40% while size and BMC increased by 200–300%. Thus, growth builds a bigger but only slightly denser skeleton. We speculate that effect of disease or a risk factor during growth depends on the regions maturational stage at the time of exposure. The earlier growth of a regions size than mass, and the differing growth patterns from region to region, predispose to site-specific deficits in bone size, vBMD, or both. Regions further from their peak may be more severely affected by illness than those nearer completion of growth. Bone fragility in old age is likely to have its foundations partly established during growth.
Functional Characterization of Osteoblasts and Osteoclasts from Alkaline Phosphatase Knockout Mice
Authors: Wennberg Charlotte , Hessle Lovisa , Lundberg Pernilla , Mauro Sonia , Narisawa Sonoko , Lerner Ulf H. , Millán José Luis
Published: April 2006

Tissue nonspecific alkaline phosphatase (TNAP) knockout (ko) mice manifest defects in bone mineralization that mimic the phenotypic abnormalities of infantile hypophosphatasia. In this article, we have searched for phenotypic differences between calvarial osteoblasts and osteoclasts in wild-type (wt), heterozygous and homozygous TNAP null mice. In vitro release of45Ca from calvarial bones, with and without stimulation with parathyroid hormone (PTH), revealed no functional difference between osteoclasts from the three TNAP genotypes. Studies of primary cultures of TNAP+/+, TNAP+/−, and TNAP−/− calvarial osteoblasts revealed no differences in the rate of protein synthesis or in the expression levels of messenger RNAs (mRNAs) for osteopontin (OP), osteocalcin (OC), collagen type I, core binding factor α1 (Cbfa 1), N-cadherin, Smad 5, and Smad 7. Release of interleukin-6 (IL-6) from calvarial osteoblasts under basal conditions and after stimulation with PTH, tumor necrosis factor α (TNF-α) or IL-1β was similar in all genotypes. The amount of cyclic adenosine monophosphate (cAMP) accumulation also was comparable. However, although cultures of primary TNAP−/− osteoblasts were able to form cellular nodules as well as TNAP positive osteoblasts do, they lacked the ability to mineralize these nodules in vitro. Mineralization also was delayed in TNAP+/− osteoblast cultures compared with cultures of wt osteoblasts. Incubation with media supplemented with recombinant TNAP, but not with enzymatically inactive TNAP, restored mineralization in ko osteoblast cultures. Our data provide evidence that osteoblasts in TNAP null mice differentiate normally but are unable to initiate mineralization in vitro. The fact that even heterozygous osteoblasts show delayed mineralization provides a rationale for the presence of bone disease in carriers of hypophosphatasia.
Further Evidence for Genetic Heterogeneity Within Type II Autosomal Dominant Osteopetrosis
Authors: Bénichou O. D. , Bénichou B. , Copin H. , De Vernejoul M. C. , Van Hul W.
Published: April 2006

Type II autosomal dominant osteopetrosis (ADO II) is characterized by an increased bone mass that contrasts with the high frequency of fractures. Linkage analysis performed in an extensive Danish family recently provided evidence for the mapping of an ADO II gene to an 8.5-cM region in chromosome 1p21 between microsatellite markers D1S486 and D1S2792. We recruited, phenotyped, and haplotyped 4F catheter ADO II families including 18 affected subjects and 29 unaffected subjects in order to narrow the candidate region and to search for genetic heterogeneity. ADO II diagnosis was ascertained by the observation of vertebral end plate thickening in at least 2 patients from successive generations. Linkage studies involved five microsatellite markers (D1S486, D1S206, D1S495, D1S248, and D1S2792) spanning 1p21. Haplotype analyses of two of our families clearly excluded the tested locus. The two remaining families gave poorly informative results. These results, combined with those previously reported in two American families, suggest that chromosomal region 1p21 is most likely a minor locus for ADO II.
Mineral Density and Bone Strength Are Dissociated in Long Bones of Rat Osteopetrotic Mutations
Authors: Tuukkanen J. , Koivukangas A. , Jämsä T. , Sundquist K. , MacKay C. A. , Marks S. C.
Published: April 2006

Bone mineral density (BMD) and mechanical strength generally show strong positive correlations. However, osteopetrosis is a metabolic bone disease with increased skeletal density radiographically and increased risk of fracture. We have evaluated mechanical strength and mineral density in three osteopetrotic mutations in the rat (incisors-absent [ia/ia], osteopetrosis [op/op], and toothless [tl/tl]) to test the hypothesis that reduced bone resorption in one or more of these mutations results in weaker bones in the presence of greater mineral density and skeletal mass. Peripheral quantitative computed tomography (pQCT) was used to analyze BMD and cross-sectional geometry in the tibial diaphysis and metaphysis as well as the femoral diaphysis and femoral neck. The bending breaking force of tibial and femoral midshafts was obtained using the three-point bending test and femoral neck strength was tested by axial loading. Osteopetrotic mutants were significantly smaller than their normal littermates (NLMs) in each stock. The pQCT analysis showed that BMD and bone mineral content (BMC) were higher than or equal to NLMs in all skeletal sites measured in the osteopetrotic mutants. However, the mechanical breaking force was equal to or lower than their NLMs in all sites. The cross-sectional structure of long bone shafts was markedly different in osteopetrotic mutants, having a thin cortex and a medullary area filled with primary trabecular bone. These results indicate that osteopetrotic mutations in the rat increase bone density and decrease bone strength. The tibial diaphysis was significantly weaker in tl/tl and ia/ia mutants and the tibial metaphysis showed the greatest increase in BMD in all mutants. These data are another illustration that an increased BMD does not necessarily lead to stronger bones.
Intercellular Adhesion Molecule 1 Discriminates Functionally Different Populations of Human Osteoblasts: Characteristic Involvement of Cell Cycle Regulators
Authors: Tanaka Yoshiya , Maruo Akihiko , Fujii Koichi , Nomi Masashi , Nakamura Toshitaka , Eto Sumiya , Minami Yasuhiro
Published: April 2006

The concept of differential regulation of certain adhesion molecules on different cell subsets and their relevance to cell functions has emerged in recent years. The initial event in bone remodeling is an increase in osteoclastic bone resorption and cell adhesion between osteoclastic precursors and bone marrow stromal cells or osteoblasts is known to commit the osteoclast development. Here, we show that human osteoblasts can be divided into two subsets based on the expression of the intercellular adhesion molecule (ICAM)-1; ICAM-1+ osteoblasts highly adhered to monocytes, including osteoclast precursors, produced osteoclast differentiation factor (ODF), and induced multinuclear osteoclast-like cell formation. Anti-ODF monoclonal antibody (mAb) did not inhibit the adhesion of monocytes to osteoblastic cells, whereas anti-leukocyte function-associated antigen (LFA)-1, a receptor for ICAM-1, mAb blocked the adhesion. We thereby propose that the higher affinity adhesion via LFA-1/ICAM-1 is prerequisite for efficient function of membrane-bound ODF during osteoclast maturation. The functional characteristics of ICAM-1+ osteoblasts were emphasized further by cell cycle regulation, as manifested by (i) up-regulation of p53 and p21, (ii) reduction of activity of cyclin-dependent kinase (cdk) 6, (iii) underphosphorylation of retinoblastoma protein, (iv) increased Fas but reduced bcl-2 expression, and (v) majority of cells remained at G0/G1 phase. Furthermore, ICAM-1+ osteoblasts were induced by interleukin-1β (IL-1β). Taken together, we propose that the differentiation of osteoblasts to ICAM-1+ subpopulation by inflammatory cytokines plays an important role in osteoporosis, which is observed in patients with chronic inflammation, because ICAM-1+ osteoblasts can bias bone turnover to bone resorption, committing osteoclast maturation through cell adhesion with its precursor, and the majority of ICAM-1+ osteoblasts arrested at G0/G1 phase. Such regulation of cell cycle arrest also is an important determinant of the life span of cells in bone in which continuous bone remodeling maintains its homeostasis.
Association of the Vitamin D Receptor Genotype BB with Low Bone Density in Hyperthyroidism
Authors: Obermayer-Pietsch Barbara M. , Frühauf Gerwig E. , Chararas Kornelia , Mikhail-Reinisch Sabine , Renner Wilfried , Berghold Andrea , Kenner Lukas , Lackner Carolin
Published: April 2006

Bone mineral density (BMD) is modulated by genetic and environmental factors or certain diseases. In several conditions such as low calcium intake, an influence of vitamin D receptor (VDR) polymorphisms on BMD has been suggested. In the present study, we investigated the relationship of Bsm I and Fok I polymorphisms of the VDR gene and BMD in patients with hyperthyroidism, a disease that often results in low BMD. Bsm I and Fok I genotypes were determined in 76 postmenopausal hyperthyroid patients and 62 healthy postmenopausal women as controls. Patients and controls were matched for age, time since menopause, and lifestyle factors and were free of estrogen medication. BMD evaluation included axial dual X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (PQCT). Low BMD was defined as −2.5 STD below the young adult mean value. Biochemical parameters investigated were thyroid hormones, osteocalcin, and 25-(OH)-vitamin D3 as well as routine laboratory data. Low BMD was found in 61% of hyperthyroid patients and in only 23% of euthyroid controls. In the group of hyperthyroid patients with low bone density, the BB genotype (VDR Bsm I polymorphisms) was significantly more frequent (39%) than in controls (13%; p = 0.003) and hyperthyroid patients with normal BMD (6%; p = 0.013). The odds ratio (OR) for low BMD in patients with BB genotype was 5.7 (95% CI, 1.7-19.1; p < 0.005) as compared with the Bb and bb genotypes and 5.5 (95% CI, 2.3-13.2; p < 0.0001) for hyperthyroidism alone. The cumulative risk for low BMD in patients with hyperthyroidism and BB genotype was 31.4 (95% CI, 3.9-256; p < 0.0003). VDR Fok I genotypes showed no significant relationship with BMD or other general or bone-specific parameters. Thus, hyperthyroidism and the genetic background of a BB genotype may promote synergistically the development of low BMD in hyperthyroid patients. Screening for the BB genotype in these patients therefore could help to identify those with particularly high risk for the development of low BMD and allow early treatment.
Characterization of Perimenopausal Bone Loss: A Prospective Study
Authors: Recker R. , Lappe J. , Davies K. , Heaney R.
Published: April 2006

This study characterized the change in bone mass, bone markers, pituitary/gonadal hormones, vitamin D, parathyroid hormone, and anthropometric variables in a cohort of healthy women as they passed through normal menopause. We recruited 75 women > 46 years old who had premenopausal estradiol (E2) and gonadotropin levels and regular menses. During 9.5 years of observation, 54 experienced normal menopause (PM group) and 21 remained estrogen replete (ER group). Before the beginning of the menopausal drop and after its completion, the slope of bone mass on time in the PM group was 0% for the spine, −0.61% per year for the total body, and −0.45% per year for the femoral neck. Designating these losses as “age related,” there were 0, 4.88, and 3.40% losses for spine, total body bone mineral (TBBM), and femoral neck, respectively, in the 8-year period for which the data were analyzed. Across menopause, we found a sigmoid pattern of bone loss in the PM group beginning about 2–3 years before the last menses and ending about 3–4 years after the last menses. The total estrogen-deprivation bone losses were 10.50, 7.73, and 5.30% for the spine, TBBM, and femoral neck, respectively. In the ER group, we found a 0, 0.59, and 0.93% per year loss in spine, TBBM, and femoral neck, respectively. Serum osteocalcin rose 77%, serum total alkaline phosphatase rose 34%, and urinary hydroxyproline/creatinine (Hypro/Cr) ratio rose 44% in the PM group, while remaining stable in the ER group. We conclude that menopausal bone loss is a composite of loss caused by estrogen deprivation and age per se for the hip and total body, but is caused by estrogen deprivation alone for the spine.
Rate of Bone Loss Is Associated with Mortality in Older Women: A Prospective Study
Authors: Kado Deborah M. , Browner Warren S. , Blackwell Terri , Gore Robert , Cummings Steven R.
Published: April 2006

Older women with low bone density have an increased risk of fracture, cardiovascular disease, and mortality. However, it is not known whether this association is caused by ongoing bone loss or by lower bone mass earlier in life. To determine whether rate of bone loss is associated with total and cause-specific mortality, we prospectively studied 6046 women aged 65 years or older who had serial bone mineral density (BMD) measurements as a part of the Study of Osteoporotic Fractures. Rates (mean ± SD) of loss of BMD at the heel (for a mean of 5.7 years) and hip (for a mean of 3.5 years) were estimated. Cause-specific mortality was ascertained from death certificates and hospital records. BMD loss at the heel was 5.9 ± 6.0 mg/cm2 per year (1.5 ± 1.5%) and BMD loss at the hip was 4.1 ± 10.2 mg/cm2 per year (0.6 ± 1.4%). During an average follow-up of 3.2 years after the second measurement of BMD, 371 deaths occurred. Each SD increase in BMD loss at the hip was associated with a 1.3-fold (95% CI, 1.1-1.4) increase in total mortality, adjusted for age, baseline BMD, diabetes, hypertension, incident fractures, smoking, physical activity, health status, weight loss, and calcium use. In particular, hip BMD loss was associated with increased mortality from coronary heart disease (relative hazard [RH] = 1.3 per SD; 95% CI, 1.0-1.8) and pulmonary diseases (RH = 1.6 per SD; 95% CI, 1.1-2.5). The findings were similar for bone loss at the heel, except there was no significant association with pulmonary mortality. These results raise the possibility that bone loss may share common etiologies with coronary and pulmonary diseases.
Contribution of Organic Material to the Ion Composition of Bone
Authors: Bushinsky David A. , Gavrilov Konstantin L. , Chabala Jan M. , Levi-Setti Riccardo
Published: April 2006

Studies of bone mineral ranging from cadaveric analysis to the use of high-resolution ion microprobe with secondary ion mass spectroscopy (SIMS) have concluded that bone is rich in sodium and potassium relative to calcium. Exposure of bone to acid conditions either in vitro or in vivo leads to an exchange of hydrogen ions for sodium and potassium buffering the acidity of the medium or blood, respectively. Whether these monovalent ions reside within the mineral or organic phases of bone has never been determined. To determine the contribution of organic material to bone ion composition, we dissected calvariae from 4- to 6-day-old mice, removed organic material of some with hydrazine (Hydr), and prepared all bones for analysis using a high-resolution scanning ion microprobe coupled to a secondary ion mass spectrometer. We found that in non-Hydr-treated calvariae (Ctl) there was far more surface sodium and potassium than calcium (23Na/40Ca = 15.7 + 1.9, ratio of counts of detected secondary ions, mean + 95% CI,39K/40Ca = 44.0 + 1.5). Removal of organic material with hydrazine (Hydr) led to a marked fall in the ratio of sodium to calcium and potassium to calcium (23Na/40Ca = 5.9 + 1.4, p < 0.025 vs. respective Ctl and39K/40Ca = 1.1 + 1.5, p < 0.001 vs. respective Ctl). Similarly, when examining the cross-section of the calvariae there was more sodium and potassium than calcium (23Na/40Ca = 8.6 + 1.6,39K/40Ca = 26.7 + 1.8). Treatment with Hydr again caused a marked fall in both ratios (23Na/40Ca = 0.3 + 1.6, p < 0.001 vs. respective Ctl and39K/40Ca = 0.02 + 1.9, p < 0.001 vs. respective Ctl). Thus, within bone the organic material contains the majority of the sodium and potassium. This suggests that the organic material in bone and not the mineral itself is responsible for the acute buffering of the additional hydrogen ions during metabolic acidosis.
Intravenous Pamidronate for Treatment of Reflex Sympathetic Dystrophy During Breast Feeding
Authors: Siminoski K. , Fitzgerald A. A. , Flesch G. , Gross M. S.
Published: April 2006

A 39-year-old woman presented in the first month of pregnancy with reflex sympathetic dystrophy involving both lower legs. Symptoms became so severe that she could not walk unassisted, and the pain worsened after delivery. Radiographs showed patchy reduction in apparent density in the tarsal bones and around the ankles and knees. Uptake was increased in these areas on technetium methylene diphosphonate bone scan. Bone density (dual-energy X-ray absorptiometry) was reduced in the spine, hip, and radius. Biochemical tests were normal except for an increase in urinary excretion of the N-telopeptide cross-linking region of type I collagen (NTx). Because the patient wanted to continue breast-feeding, intravenous pamidronate was administered at monthly intervals. Breast milk was collected for 48 h after the infusion. The pain began to decrease soon after drug administration was initiated, and it was virtually gone by 6 months. NTx excretion fell by 78% and bone density increased by as much as 18.9% over the 6-month treatment interval. The baby was healthy and grew normally. Milk expressed after the first treatment was assayed for pamidronate content by high-performance liquid chromatography with fluorescence detection. None was detected (limit of quantitation, 0.4 μmol/liter). This case shows that pamidronate may be considered for treatment of lactating women.