bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence of Low Femoral Bone Density in Older U.S. Adults from NHANES III
Authors: Looker Anne C. , Orwoll Eric S. , Johnston C. Conrad , Lindsay Robert L. , Wahner Heinz W. , Dunn William L. , Calvo Mona S. , Harris Tamara B. , Heyse Stephen P.
Published: April 2006

Most estimates of osteoporosis in older U.S. adults have been based on its occurrence in white women, even though it is known to affect men and minority women. In the present study, we used dual-energy X-ray absorptiometry measurements of femoral bone mineral density (BMD) from the third National Health and Nutrition Examination Survey (NHANES III, 1988–1994) to estimate the overall scope of the disease in the older U.S. population. Specifically, we estimate prevalences of low femoral BMD in women 50 years and older and explore different approaches for defining low BMD in older men in that age range. Low BMD levels were defined in accordance with an approach proposed by an expert panel of the World Health Organization and used BMD data from 382 non-Hispanic white (NHW) men or 409 NHW women ages 20–29 years from the NHANES III dataset. For women, estimates indicate 13–18%, or 4–6 million, have osteoporosis (i.e., BMD >2.5 standard deviations [SD] below the mean of young NHW women) and 37–50%, or 13–17 million, have osteopenia (BMD between 1 and 2.5 SD below the mean of young NHW women). For men, these numbers depend on the gender of the reference group used to define cutoff values. When based on male cutoffs, 3–6% (1–2 million) of men have osteoporosis and 28–47% (8–13 million) have osteopenia; when based on female cutoffs, 1–4% (280,000–1 million) have osteoporosis and 15–33% (4–9 million) have osteopenia. Most of the older U.S. adults with low femur BMD are women, but, regardless of which cutoffs are used, the number of men is substantial.
Expression of the CT/CGRP Gene and Its Regulation by Dibutyryl Cyclic Adenosine Monophosphate in Human Osteoblastic Cells
Authors: Drissi H. , Hott M. , Marie P. J. , Lasmoles F.
Published: April 2006

There is general agreement that calcitonin (CT) inhibits bone resorption by its effects on osteoclast function. CT was also found to have direct effects on osteoblast-like cells. In this study, we investigated the expression of CT and calcitonin gene-related peptide (CGRP), the two peptides encoded by the CT/CGRP gene, in human osteosarcoma cell lines and in normal human trabecular osteoblastic cells (HOB), and we studied the modulation of CT/CGRP gene expression by dibutyryl cyclic adenosine monophosphate ((Bu)2 cAMP), a cAMP analog. We first detected by Northern blot hybridization the presence of CT and CGRP mRNAs in different osteosarcoma cell lines (OHS-4, MG-63, Saos-2, HOS-TE85) and HOB cells. In the steady state, OHS-4 cells express slightly more CT and CGRP mRNAs than other cell lines or normal human osteoblasts, in parallel with messengers of differentiated osteoblasts, such as osteocalcin (OC) and alkaline phosphatase (ALP). OHS-4 cells also express CT and CGRP proteins, as demonstrated by immunocytochemistry. Stimulation of OHS-4 cells with 1 mM (Bu)2 cAMP induced a significant increase in mRNA levels for CT (×2.5) and CGRP (×3), as determined by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) procedure. The involvement of a transcriptional mechanism in this effect was evidenced by nuclear run-off transcription assay. In addition, (Bu)2 cAMP increased OC (×4) and ALP (×3) mRNA levels in OHS-4 cells. These effects were observed at 24 h and were maximal at 48 h, indicating that (Bu)2 cAMP induced cell differentiation and increased the transcription of the CT/CGRP gene in OHS-4 osteoblast-like cells. The results indicate that human osteosarcoma cells and primary human osteoblastic cells express CT and CGRP mRNA and proteins, and that (Bu)2 cAMP, an activator of protein kinase A, induces up-regulation of osteoblastic phenotypic genes and enhances CT and CGRP gene transcription, indicating that induction of osteoblastic differentiation by (Bu)2 cAMP is associated with enhanced expression of CT and CGRP in human osteoblastic cells.
Premenopausal Ovariectomy-Related Bone Loss: A Randomized, Double-Blind, One-Year Trial of Conjugated Estrogen or Medroxyprogesterone Acetate
Authors: Prior Jerilynn C. , Vigna Yvette M. , Wark John D. , Eyre David R. , Lentle Brian C. , Li David K. B. , Ebeling Peter R. , Atley Lynne
Published: April 2006

The purpose of this study was to contrast the effects of conventional estrogen treatment with medroxyprogesterone on cancellous and cortical bone change in the first year following premenopausal ovariectomy. This 1-year double-blind randomized therapy trial was stratified by osteoporosis family history and performed in an academic medical center and community hospitals. Premenopausal women 45 ± 5 years old, postovariectomy for benign diseases were provided 600 mg/day of calcium and randomized to daily therapy with conjugated equine estrogen (CEE, 0.6 mg) or medroxyprogesterone (MPA, 10 mg). The primary outcome variable was spinal quantitative computed tomography (QCT) bone density change over 1 year with additional outcomes of dual-energy X-ray absorptiometry (DXA) of proximal femur (FN), whole body (WB), and spine segment (WBS) and N-telopeptide, bone-specific alkaline phosphatase, and other bone marker, hormonal, and weight changes. Results in the 33 women completing the study, whose initial bone densities were normal (QCT 133 mg/cm3, femoral neck 0.94 g/cm2, whole body DXA 1.13 g/cm2), showed annual QCT loss during CEE therapy of −11.5 mg/cm3 (p < 0.0007) and MPA bone loss of −19.7 mg/cm3 (p < 0.0001). Losses were marginally greater on MPA than CEE (p = 0.04). Extremely high postovariectomy (5 days) and pretreatment resorption markers (>3 SD above premenopausal normal levels) were significantly related to bone loss. Across the year, resorption decreased during CEE but increased on MPA treatment. Significant DXA bone losses were prevented by CEE treatment (−1.4% FN, −.4% WB, and −1.5% WBS, all NS). However, DXA bone loss was not prevented by MPA treatment (−5% FN, −2.8% WB, and −6.1% WBS, all p < 0.03). Average weight gain was significant (+3.2 ± 4.0 kg) and greater on CEE than MPA (+4.7 vs. +2.0 kg, p = 0.049). In conclusion, CEE therapy did not prevent significant 8% cancellous spinal bone loss in the first year following premenopausal ovariectomy, despite supplementation with 600 mg/day of calcium, good control of vasomotor symptoms, and nearly 5 kg of gain in weight. Significant DXA bone loss, however, was prevented by CEE, but not by MPA therapy. These unexpected results were statistically related to high bone resorption following ovariectomy, which CEE suppressed but MPA did not. Bone formation markers increased during MPA therapy but were unchanged during CEE therapy.
Parathyroid Hormone Enhances Early and Suppresses Late Stages of Osteogenic and Chondrogenic Development in a BMP-Dependent Mesenchymal Differentiation System (C3H10T½)
Authors: Hollnagel Angela , Ahrens Marion , Gross Gerhard
Published: April 2006

The role of parathyroid hormone (PTH) upon osteo-/chondrogenic development was investigated in a bone morphogenetic protein (BMP)-dependent differentiation system involving the recombinant expression of BMPs in mesenchymal progenitor cells (C3H10T½). The constitutive expression of the PTH/PTH related protein receptor in this system led to a marked stimulation of chondrogenic and osteogenic development, while the permanent application of the ligand PTH(1–34) resulted in opposite responses by stimulating the early and suppressing the late stages of osteo-/chondrogenic development. These contrasting effects of PTH(1–34) on osteogenic and chondrocytic development seem, therefore, to depend on the cellular state of differentiation. The osteogenic and chondrocytic differentiation potential was substantiated histologically and by genetic analyses of marker genes like c-fos, alkaline phosphatase, osteocalcin, collagen α1(I), and collagen α1(II). The capacity to regulate osteogenic and chondrogenic development is located in the amino-terminal (1–34) region of the PTH molecule and seems to be mediated by the cyclic adenosine monophosphate signaling cascade. The application of other PTH domains like PTH(28–48) and PTH(53–84) did not exhibit significant responses. PTH acts as an essential factor in mesenchymal development controlling rates of differentiation into the osteogenic or chondrogenic lineage. The analysis of PTH effects in this system demonstrates the value of recombinant mesenchymal progenitor cells in the in vitro analysis of osteo-/chondrogenic development.
Developmental Restriction of Embryonic Calvarial Cell Populations as Characterized by Their In Vitro Potential for Chondrogenic Differentiation
Authors: Toma Cyril D. , Schaffer Jonathan L. , Meazzini M. Costanza , Zurakowski David , Nah Hyun-Duck , Gerstenfeld Louis C.
Published: April 2006

The mechanism(s) by which the cells within the calvaria tissue are restricted into the osteogenic versus the chondrogenic lineage during intramembranous bone formation were examined. Cells were obtained from 12-day chicken embryo calvariae after tissue condensation, but before extensive osteogenic differentiation, and from 17-day embryo calvariae when osteogenesis is well progressed. Only cell populations from the younger embryos showed chondrogenic differentiation as characterized by the expression of collagen type II. The chondrocytes underwent a temporal progression of maturation and endochondral development, demonstrated by the expression of collagen type II B transcript and expression of collagen type X mRNA. Cell populations from both ages of embryos showed progressive osteogenic differentiation, based on the expression of osteopontin, bone sialoprotein, and osteocalcin mRNAs. Analysis using lineage markers for either chondrocytes or osteoblasts demonstrated that when the younger embryonic cultures were grown in conditions that were permissive for chondrogenesis, the number of chondrogenic cells increased from ∼15 to ∼50% of the population, while the number of osteogenic cells remained almost constant at ∼35–40%. Pulse labeling of the cultures with BrdU showed selective labeling of the chondrogenic cells in comparison with the osteogenic cells. These data indicate that the developmental restriction of skeletal cells of the calvaria is not a result of positive selection for osteogenic differentiation but a negative selection against the progressive growth of chondrogenic cells in the absence of a permissive or inductive environment. These results further demonstrate that while extrinsic environmental factors can modulate the lineage progression of skeletal cells within the calvariae, there is a progressive restriction during embryogenesis in the number of cells within the calvaria with a chondrogenic potential. Finally, these data suggest that the loss of cells with chondrogenic potential from the calvaria may be related to the progressive limitation of the reparative capacity of the cranial bones.
Regulation of Interleukin-6 Secretion from Mononuclear Blood Cells by Extracellular Calcium
Authors: Bornefalk Eva , Ljunghall Sverker , Lindh Erik , Bengtson Olof , Johansson Anna G. , Ljunggren Östen
Published: April 2006

Interleukin-6 (IL-6) is known to enhance osteoclast recruitment, and thereby bone resorption. Thus, IL-6 has been proposed to mediate hypercalcemia in multiple myeloma and the enhanced osteoclastic activity seen in postmenopausal osteoporosis. We recently reported that the calcium concentration in plasma affects IL-6 secretion from mononuclear blood cells. To investigate the underlying mechanism, we have studied the effect of calcium on IL-6 formation in mononuclear blood cells ex vivo and in vitro. Thirteen healthy volunteers were given 1 g of calcium orally after overnight fasting. Plasma levels of ionized calcium (pCa2+) and serum levels of parathyroid hormone (sPTH) were measured after 2 and 4 h, with all subjects still fasting. After 2 h, pCa2+ was increased and sPTH decreased in all 13 persons. IL-6 secretion ex vivo from mononuclear blood cells drawn 4 h after calcium intake was increased 185% as compared with IL-6 secretion from cells drawn just before calcium intake. In control experiments without calcium intake, there was no alteration in pCa2+ and no effect on IL-6 secretion from mononuclear blood cells. In vitro studies revealed that stimulation of isolated mononuclear blood cells with physiological concentrations of calcium dose-dependently increased IL-6 secretion with an estimated EC50 at 1.2 mM Ca2+. No effect on the IL-6 secretion was seen following treatment of the isolated mononuclear blood cells with PTH or calcitonin. These observations demonstrate that the plasma calcium concentration affects IL-6 secretion from mononuclear blood cells. The in vitro data indicate the involvement of a direct calcium sensing mechanism. These findings might have implications in hypercalcemia and should also be borne in mind when considering the role of cytokines in osteoporosis.
Reduced Rates of Skeletal Remodeling Are Associated with Increased Bone Mineral Density During the Development of Peak Skeletal Mass
Authors: Slemenda Charles W. , Peacock Munro , Hui Siu , Zhou Lifen , Johnston C. Conrad
Published: April 2006

Two related studies were conducted to assess the associations between markers of skeletal modeling and remodeling in healthy children. Members of monozygotic twin pairs, aged 6–14, enrolled in a clinical trial of calcium supplementation, were studied at the end of the period of supplementation and for 3 years thereafter. Supplemented children had significantly higher rates of gain in bone mineral density (BMD) (+3% on average) during the period of supplementation accompanied by significantly lower concentrations of serum osteocalcin (OC, −15%). During postsupplement follow-up, both differences in BMD and OC disappeared. Black females, age matched to the baseline ages of the white children, had significantly lower serum concentrations of both OC and tartrate-resistant acid phosphatase (TRAP) at all ages and higher BMDs. When stratified on serum TRAP concentrations, regardless of race, children with lower concentrations had significantly higher BMDs, and no racial differences were apparent. In regression models accounting for 70–80% of the variability in BMD in children, body size and TRAP, but not race, remained significantly associated with BMD. The skeletal advantages seen with calcium supplementation and black race appear to be associated with reduced rates of skeletal turnover. Given that markers of turnover during growth reflect both skeletal modeling and remodeling, and there is no apparent advantage to reduced skeletal modeling, it seems probable that reduced remodeling is the factor that accounts for the increases in bone mass.
The Ca2+-Sensing Receptor (CaR) Activates Phospholipases C, A2, and D in Bovine Parathyroid and CaR-Transfected, Human Embryonic Kidney (HEK293) Cells
Authors: Kifor Olga , Diaz Ruben , Butters Robert , Brown Edward M.
Published: April 2006

The extracellular Ca2+ (Cao2+)-sensing receptor (CaR) is a G protein–coupled receptor that activates phospholipase C (PLC). In the present studies, we assessed Cao2+-dependent changes in the generation of inositol phosphates (IP), free arachidonic acid (AA), and phosphatidylbutanol (PtdBtOH) by PLC, phospholipase A2 (PLA2), and phospholipase D (PLD), respectively, in bovine parathyroid cells as well as in wild-type or CaR-transfected human embryonic kidney (HEK293) cells (HEK-WT and HEK-CaR, respectively). Elevated Cao2+ increased the formation of IPs in parathyroid cells as well in HEK-CaR but not in HEK-WT cells. High Cao2+ also elicited time- and dose-dependent increases in PtdBtOH in parathyroid cells and HEK-CaR but not in HEK-WT cells. Brief treatment of parathyroid and HEK-CaR cells with an activator of protein kinase C (PKC), phorbol 12-myristate,13-acetate (PMA), stimulated PLD activity at both low and high Cao2+. Moreover, high Cao2+-stimulated PLD activity was abolished following down-regulation of PKC by overnight phorbol myristate acetate (PMA) pretreatment, suggesting that CaR-mediated activation of PLD depends largely upon stimulation of PKC. High Cao2+ likewise increased the release of free AA in parathyroid and HEK-CaR but not in HEK-WT cells. Mepacrine, a general PLA2 inhibitor, and AACOCF3, an inhibitor of cytosolic PLA2, reduced AA release in parathyroid cells at high Cao2+, suggesting a major role for PLA2 in high Cao2+-elicited AA release. Pretreatment of parathyroid cells with PMA stimulated release of AA at low and high Cao2+, while a PKC inhibitor, chelerythrine, reduced AA release at high Cao2+ to the level observed with low Cao2+ alone. Thus, PKC contributes importantly to the high Cao2+-evoked, CaR-mediated activation of not only PLD but also PLA2. Finally, high Cao2+-stimulated production of IP, PtdBtOH, and AA all decreased substantially in parathyroid cells cultured for 4 days, in which expression of the CaR decreases by 80% or more, consistent with mediation of these effects by the receptor. Thus, the CaR activates, directly or indirectly, at least three phospholipases in bovine parathyroid and CaR-transfected HEK293 cells, providing for coordinate, receptor-mediated regulation of multiple signal transduction pathways in parathyroid and presumably other CaR-expressing cells.
Evidence of a Correlation of Estrogen Receptor Level and Avian Osteoclast Estrogen Responsiveness
Authors: Pederson Larry , Kremer Matthew , Foged Niels Tækker , Winding Bent , Ritchie Candace , Fitzpatrick Lorraine A. , Oursler Merry Jo
Published: April 2006

Isolated osteoclasts from 5-week-old chickens respond to estradiol treatment in vitro with decreased resorption activity, increased nuclear proto-oncogene expression, and decreased lysosomal enzyme secretion. This study examines osteoclasts from embryonic chickens and egg-laying hens for evidence of estrogen responsiveness. Although osteoclasts from both of these sources express estrogen receptor mRNA and protein, estradiol treatment had no effect on resorption activity. In contrast to the lack of effect on resorption, estradiol treatment for 30 minutes resulted in steady-state mRNA levels of c-fos and c-jun increasing in osteoclasts from embryonic chickens and decreasing in osteoclasts from egg-laying hens. These data suggest that a nuclear proto-oncogene response may not be involved in estradiol-mediated decreased osteoclast resorption activity. To examine the influence of circulating estrogen on osteoclast estrogen responsiveness, 5-week-old chickens were injected with estrogen for 4 days prior to sacrifice. Estradiol treatment of osteoclasts from these chickens did not decrease resorption activity in vitro. Transfection of an estrogen receptor expression vector into osteoclasts from the estradiol-injected chickens and egg-laying hens restored estrogen responsiveness. Osteoclasts from 5-week-old chickens and estradiol treated 5-week-old chickens transfected with the estrogen receptor expression vector contained significantly higher levels of estrogen receptor protein and responded to estradiol treatment by decreasing secretion of cathepsins B and L and tartrate-resistant acid phosphatase. In contrast, osteoclasts from embryonic chickens, egg-laying hens, and estradiol-treated 5-week-old chickens either untransfected or transfected with an empty expression vector did not respond similarly. These data suggest that modulation of osteoclast estrogen responsiveness may be controlled by changes in the osteoclast estrogen receptor levels.
Modulation of the Effects of Glucocorticoids on Collagen Synthesis in Fetal Rat Calvariae by Insulin-like Growth Factor Binding Protein-2
Authors: Kream Barbara E. , Tetradis Sotirios , Lafrancis Denise , Fall Pamela M. , Feyen Jean H. M. , Raisz Lawrence G.
Published: April 2006

To test the hypothesis that insulin-like growth factors (IGFs) play a role in the response of bone to glucocorticoids, we determined the effects of cortisol on the incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP), the percent collagen synthesis, and the incorporation of [3H]thymidine into DNA of 21-day fetal rat calvariae cultured in the presence and absence of recombinant human insulin-like growth factor binding protein-2 (IGFBP-2). At 24 h, cortisol (100 nM) increased CDP labeling and the percent collagen synthesis, and these effects were blocked by IGFBP-2 (1000 nM). At 24 h, cortisol decreased the incorporation of [3H]thymidine into bone, which was not affected by the addition of IGFBP-2. At 48 h, cortisol (1000 nM) decreased CDP labeling, which was maintained in the presence of IGFBP-2. At 48 h, IGFBP-2 alone decreased basal levels of CDP and NCP labeling and the percent collagen synthesis. Our data suggest that endogenous IGFs maintain basal levels of collagen synthesis and mediate the early stimulatory effect of glucocorticoids on collagen synthesis in fetal rat calvariae. However, blocking endogenous IGFs does not abrogate the inhibitory effect of glucocorticoids on DNA synthesis and the later inhibition of collagen synthesis in calvariae.
Measurement of Bone Degradation Products in Serum Using Antibodies Reactive with an Isomerized Form of an 8 Amino Acid Sequence of the C-Telopeptide of Type I Collagen
Authors: Bonde Martin , Garnero Patrick , Fledelius Christian , Qvist Per , Delmas Pierre D. , Christiansen Claus
Published: April 2006

An enzyme-linked immunosorbent assay for measuring type I collagen degradation products in serum (S-ELISA) was developed. The assay uses a high affinity polyclonal antibody which reacts with an isomerized form of an 8 amino acid sequence of the C-telopeptides of type I collagen (EKAHD-β-GGR). Cross-reactivity to a nonisomerized synthetic peptide form of the 8 amino acid sequence is less than 0.2%. Values obtained in a group of premenopausal women (age, 33.3 ± 3.11 years) were 69 ± 24 ng/ml (n = 22). In a group of early postmenopausal women (age, 51.8 ± 1.88 years) values obtained were 125 ± 43 ng/ml (n = 46), which represents an increase of 81% (p < 0.001). Values found in untreated patients with Paget's disease were 234 ± 95 ng/ml (n = 15), and for primary hyperparathyroidism we found 335 ± 82 ng/ml (n = 10). Interveneous administration of a bisphosphonate (Pamidronate) to Paget's disease patients for 3 days was reflected in the S-ELISA by a decrease in the values of 55% when compared with values before treatment (n = 15). Following treatment with another bisphosphonate (Alendronate) for 6 months, values were decreased to 48 ± 19 ng/ml (n = 12), which corresponds to a 62% decrease. Clinical results presented in this context support that the assay is a sensitive and specific index of bone resorption. It may, therefore, prove useful in the follow up of treatment of patients with metabolic bone diseases and in the clinical investigation of osteoporosis.
Linkage Studies of a Missouri Kindred with Autosomal Dominant Spondyloepimetaphyseal Dysplasia (SEMD) Indicate Genetic Heterogeneity
Authors: Gertner Joseph M. , Whyte Michael P. , Dixon Peter H. , Pang Joanna T. , Trump Dorothy , Pearce Simon H. S. , Wooding Carol , Thakker Rajesh V.
Published: April 2006

A four-generation kindred (14 affected and 10 unaffected members) from Missouri, U.S.A. in which spondyloepimetaphyseal dysplasia (SEMD) had been inherited as an autosomal dominant disorder was investigated for linkage to 13 candidate loci: COL2AI, COL9AI, COL9A2, COL9A3, COL10A1, COL11A1, COL11A2, PSACH, FGFR3, decorin, CRTL1, COMP, and PTHRP. Mutations of COL2A1, COL9A2, COL10, and FGFR3 have been reported previously in the Strudwick type of SEMD, multiple epiphyseal dysplasia type 2 (EDM2), the Schmid type of metaphyseal dysplasia, and in achondroplasia, respectively, and the pseudoachondroplasia (PSACH) locus has been mapped to chromosome 19p12. In addition, mutations in COL9 and COL11A are associated with murine forms of degenerative joint disease and chondroplasia, respectively. The family proved informative for 12 of the 13 loci and was uninformative at the decorin locus. Linkage between this form of SEMD, designated the Missouri variant, SEMDMO, and the 12 informative candidate loci was excluded (LOD scores <−2.00 at θ = 0.005 to 0.15), thereby indicating further genetic heterogeneity in these inherited disorders of bone and cartilage development.
Cortical Remodeling Following Suppression of Endogenous Estrogen with Analogs of Gonadotrophin Releasing Hormone
Authors: Bell Karen L. , Loveridge Nigel , Lindsay Peter C. , Lunt Mark , Garrahan Nigel , Compston Juliet E. , Reeve Jonathan
Published: April 2006

The effects of estrogen suppression on osteonal remodeling in young women was investigated using transiliac biopsies (eight paired biopsies + four single pre; three single post biopsies) taken before and after treatment for endometriosis (6 months) with analogs of gonadotrophin releasing hormone (GnRH). Estrogen withdrawal increased the proportion of Haversian canals with an eroded surface (106%, p = 0.047), a double label (238%, p = 0.004), osteoid (71%, p = 0.002), and alkaline phosphatase (ALP) (116%, p = 0.043) but not those showing tartrate-resistant acid phosphatase (TRAP) activity (p = 0.25) or a single label (p = 0.30). Estrogen withdrawal increased TRAP activity in individual osteoclasts in canals with diameters greater than 50 μm (p = 0.0089) and also the number of osteons with diameters over 250 μm (p = 0.049). ALP activity in individual osteoblasts was increased but not significantly following treatment (p = 0.051). Wall thickness was significantly correlated with osteon diameter (p < 0.001). In a separate group of patients (four pairs + one post biopsy) on concurrent treatment with tibolone, there was no significant increase in the osteon density, cortical porosity, median canal diameter, or the markers of bone formation and resorption. Enzyme activities and numbers of active canals were also not increased with the concurrent treatment, but there was still an increase in the osteon diameter. As previously shown for cancellous bone, estrogen withdrawal increased cortical bone turnover. We have now shown that resorption depth within Haversian systems was also increased with treatment. The enhanced TRAP activity in individual osteoclasts supports the concept that osteoclasts are more active following estrogen withdrawal in agreement with theoretical arguments advanced previously. Understanding the cellular and biochemical mechanisms responsible for increased depth of osteoclast resorption when estrogen is withdrawn may allow the development of new strategies for preventing postmenopausal bone loss.
Serum Levels of Insulin-like Growth Factor (IGF) I, IGF-Binding Protein (IGFBP)-2, and IGFBP-3 in Osteoporotic Patients with and without Spinal Fractures
Authors: Sugimoto Toshitsugu , Nishiyama Katsuhito , Kuribayashi Fumihiko , Chihara Kazuo
Published: April 2006

The present study was performed to investigate the role of insulin-like growth factor I (IGF-I), IGF-binding protein-2 (IGFBP-2), and IGFBP-3 in age-dependent bone loss in postmenopausal Japanese women. One hundred and sixty-five Japanese women aged 43–88 years (mean age, 62) were enrolled in the cross-sectional study. Bone mineral density (BMD) was measured at the lumbar spine, femoral neck, and midradius by dual-energy X-ray absorptiometry or single-photon absorptiometry. Serum levels of IGF-I, IGFBP-2, and IGFBP-3 were measured by radioimmunoassay. BMD at all sites as well as serum levels of IGF-I and IGFBP-3 declined with age, while the serum IGFBP-2 level increased with age. Serum IGFBP-3 and −2 levels were positively and negatively correlated with the serum IGF-I level, respectively. Serum IGF-I and IGFBP-3 levels showed positive correlationship with BMD at any site, particularly at the midradius, while the serum IGFBP-2 level showed negative correlation with BMD. Multiple regression analyses showed age-independent positive correlation between the serum IGF-I level and BMD at all sites as well as age-independent positive correlation between the serum IGFBP-3 level and midradius BMD. The relationship between susceptibility to osteoporotic spinal fracture and serum IGF-I, IGFBP-3, or −2 levels was examined by decade to exclude the influence of aging. Serum levels of IGF-I and IGFBP-3 were significantly lower in subjects with spinal fractures than those without fractures at any decade. No significant difference of serum IGFBP-2 level was observed between subjects with and without fractures. The present findings suggest that IGF-I and IGFBP-3 are important to maintaining bone mass quantitatively as well as qualitatively, and that the determination of serum IGF-I and IGFBP-3 levels could be clinically useful to predict the severity of osteoporosis, particularly the risk of bone fracture associated with osteoporosis.
Precision and Discriminatory Ability of Calcaneal Bone Assessment Technologies
Authors: Greenspan Susan L. , Bouxsein Mary L. , Melton Mary E. , Kolodny Andrea H. , Clair James H. , Delucca Paul T. , Stek Michael , Faulkner Kenneth G. , Orwoll Eric S.
Published: April 2006

To determine if measuring skeletal status at the calcaneus is a potentially valuable technique for diagnosing osteoporosis, we examined five calcaneal assessment techniques in 53 young normal women and 108 postmenopausal women with osteoporosis and compared these measurements to dual-energy X-ray absorptiometry (DEXA) at the calcaneus, hip, and spine. The five instruments, including single-energy X-ray absorptiometry (SEXA) and four quantitative ultrasound (QUS) instruments, were evaluated for precision, ability to discriminate osteoporotic from young normal subjects, and correlation to the other instruments. The coefficient of variation (%CV) for instrument, positioning, interobserver, and short-term precision of the five calcaneal instruments ranged from 1.34–7.76%, 1.63–7.00%, 1.84–9.44%, and 1.99–7.04%, respectively. The %CVs for positioning, interobserver, and short-term precision were similar for calcaneal DEXA, calcaneal SEXA, and stiffness (as measured by Achilles). The %CVs for instrument precision were similar between calcaneal DEXA and SEXA. The ability of the five calcaneal instruments to discriminate osteoporotic from young normal subjects was similar based on the analysis of area under the receiver operating characteristic curves (range 0.88–0.93) and equivalent to DEXA of the calcaneus and hip (0.88–0.93). The correlations between the measurements of five calcaneal instruments were strong (0.80 ≤ r ≤ 0.91, p < 0.001). These data suggest that although the precision is variable, the calcaneal QUS and SEXA instruments can discriminate between osteoporotic patients and young normal controls and appear to be a useful technique for assessment of osteoporosis.
Reduced Bone Mass in Dutch Adolescents Fed a Macrobiotic Diet in Early Life
Authors: Parsons Tessa J. , van Dusseldorp Marijke , van Der Vliet Martine , van de Werken Karen , Schaafsma Gertjan , van Staveren Wija A.
Published: April 2006

This study investigated the effect of a macrobiotic (vegan-type) diet, low in calcium and vitamin D, consumed in early life, on bone mineral during adolescence. Bone mineral content (BMC) and bone area were measured in 195 adolescents (103 girls, 92 boys) aged 9–15 years, using dual-energy X-ray absorptiometry. Ninety-three adolescents (43 girls, 50 boys) had followed a macrobiotic diet in childhood, and 102 (60 girls, 42 boys) were control subjects. After adjustment for bone area, weight, height, percent body lean, age, and puberty, BMC was significantly lower in macrobiotic subjects, in boys and girls, respectively, at the whole body, −3.4% and −2.5%, spine, −8.5% and −5.0%, femoral neck, −8.0% and −8.2%, midshaft radius, −6.8% and −5.6%, and also in girls, at the trochanter, −5.8% (p < 0.05). No group differences were observed at the wrist. Group differences were not explained by current calcium intake or physical activity. We conclude that the use of a macrobiotic diet in early childhood negatively influences adjusted bone mass at age 9–15 years, observations which may hold important implications for fracture risk in later life.
A Comparative Study of Trabecular Bone Properties in the Spine and Femur Using High Resolution MRI and CT
Authors: Link Thomas M. , Majumdar Sharmila , Lin John C. , Newitt David , Augat Peter , Ouyang Xiaolong , Mathur Ashwini , Genant Harry K.
Published: April 2006

The purpose of this study was to use high resolution (HR) magnetic resonance (MR) and computed tomography (CT) images combined with texture analysis to investigate the trabecular structure of human vertebral and femoral specimens and to compare these techniques with bone mineral density (BMD) in the prediction of bone strength. Twenty-nine bone cubes were harvested from 12 proximal femur cadaver specimens and 29 from 8 spines. HR MR and CT images were obtained, and texture analysis techniques were used to assess trabecular structure. Additionally, BMD, elastic modulus (EM), and maximum compressive strength were determined. R2 for EM versus texture measures computed in the MR images was higher (R2 = 0.27–0.64, p < 0.01) in the spine than in the femur specimens (R2 = 0.12–0.22, p < 0.05). R2 values were similar in the CT images. R2 for EM versus BMD was 0.66 (p < 0.01) in the spine and 0.61 (p < 0.01) in the femur specimens. In the MR images, texture measures combined with BMD in a multivariate-regression model significantly increased R2, while improvement was less significant in the CT images. Thus, texture analysis may provide additional information needed to analyze bone strength and quality.
Lack of Correlation Between Start Codon Polymorphism of the Vitamin D Receptor Gene and Bone Mineral Density in Premenopausal French Women: The OFELY Study
Authors: Eccleshall T. Ross , Garnero Patrick , Gross Coleman , Delmas Pierre D. , Feldman David
Published: April 2006

Previous studies have demonstrated an association between bone mineral density (BMD) and a start codon polymorphism (SCP) of the vitamin D receptor (VDR) gene in pre- and postmenopausal Caucasian and Japanese women. The SCP can be determined by a restriction fragment length polymorphism defined by the FokI restriction endonuclease. VDR alleles containing the FokI site are denoted by f and alleles lacking the site by F. In this study, the association between BMD and the SCP was examined in a group of 174 premenopausal French women who previously had been studied for a relationship between BMD and the VDR BsmI polymorphism. The SCP genotypes of the French women were FF 40%, Ff 44%, and ff 16% and they were independent of the BsmI genotype. BMD was measured by dual-energy X-ray absorptiometry at the lumbar spine, proximal femur, forearm, and total body. In contrast to previous reports, there was no association of BMD with SCP genotype in this group of Caucasian women at any site. We also measured several biochemical indices of calcium homeostasis and bone turnover. We found no statistically significant associations between SCP genotype and calcium, parathyroid hormone, or vitamin D levels. There was a 33.5% higher level of the skeletal resorption marker N-telopeptides of type I collagen in the women with the ff genotype when compared with women with the FF genotype (p = 0.004). Other bone turnover markers failed to show an association with SCP genotype. In summary, the SCP genotype may not be associated with reduced BMD in all geographical or ethnic populations.
Skeletal Effects of Low-Dose Cyclosporin A in Aged Male Rats: Lack of Relationship to Serum Testosterone Levels
Authors: Dr. Erben Reinhold G. , Stangassinger Manfred , Gärtner Roland
Published: April 2006

The aim of this study was to investigate the skeletal effects of cyclosporin A (CsA) in a dose range relevant to clinical medicine in lumbar vertebral cancellous bone of aged male rats and to correlate these effects with possible changes in serum testosterone levels. Thirty-one 18-month-old male Wistar rats were divided into four weight-matched groups and subcutaneously injected with either 0, 1, 3, or 5 mg of CsA/kg of body weight three times per week. After 4 weeks of treatment, all rats were killed after in vivo fluorochrome labeling and the first lumbar vertebrae analyzed by quantitative histomorphometry. Serum was analyzed for total calcium, creatinine, alkaline phosphatase, osteocalcin, parathyroid hormone, total testosterone, and CsA levels. CsA administration resulted in a dose-dependent increase in serum osteocalcin levels and in histomorphometric indices of cancellous bone turnover in the axial skeleton. Furthermore, CsA-treated rats showed a deterioration of vertebral cancellous bone structure with increased discontinuity of the trabecular bone network due to trabecular plate perforations. Serum testosterone levels were not significantly changed by CsA treatment and were uncorrelated to all biochemical or histomorphometric indices of bone turnover. We conclude that the 4-week administration of CsA at doses that are close to those used in transplantation patients induced high turnover osteopenia in the axial skeleton of aged, 18-month-old male rats, and that these effects were likely not mediated by changes in serum testosterone levels.
Leukemia Inhibitory Factor and Oncostatin M Influence the Mineral Phases Formed in a Murine Heterotopic Calcification Model: A Fourier Transform-Infrared Microspectroscopic Study
Authors: Bohic S. , Rohanizadeh R. , Touchais S. , Godard A. , Daculsi G. , Heymann D.
Published: April 2006

The study of bone mineralization processes is of considerable interest in understanding bone diseases and developing new therapies for skeletal disorders, particularly since bone homeostasis requires numerous cell types and a large cytokine network. Cell culture models of mineralization have often been used to study the cellular mechanisms of mineralization, but few data have been reported concerning the influence of extracellular matrix components and cytokines on the physicochemical properties of mineral. The purpose of this study was to analyze the effects of two cytokines, leukemia inhibitory factor (LIF) and oncostatin M (OSM), involved in bone metabolism on the physicochemical properties of bone mineral formed in a murine in vivo mineralization model. Murine bone marrow cells implanted under the kidney capsule in the presence or absence of cytokines led to heterotopic ossicle formation. A scanning electron microscopic microprobe revealed that heterotopic calcification had a lower (∼20%) Ca/P ratio after cytokine treatment as compared with the control without cytokine. Transmission electron microscopic analysis of cytokine-treated ossicles showed numerous areas with low mineral density, whereas electron diffraction pattern revealed an apatitic phase. These areas were not observed in the absence of cytokine. Moreover, Fourier transform-infrared microspectroscopy showed at the molecular level that the presence of either cytokine induced many microscopic areas in which short-range order organization, such as incorporation of carbonate and crystallinity/maturity of ossicle mineral, were modified. LIF and OSM influenced mineral phase formation in the present model and may thus be key protagonists in bone mineral development and skeletal diseases.
A New Method to Isolate Large Numbers of Rabbit Osteoclasts and Osteoclast-like Cells: Application to the Characterization of Serum Response Element Binding Proteins During Osteoclast Differentiation
Authors: David Jean-Pierre , Neff Lynn , Chen Yan , Rincon Mercedes , Horne William C. , Baron Roland
Published: April 2006

We have developed a new method that allows the purification of large numbers of both authentic osteoclasts (OCs) and in vitro differentiated osteoclast-like cells (OCLs) from rabbits. We characterized the OCLs in terms of the expression of different phenotypic markers of OC differentiation and their ability to resorb bone. The method provides a system for performing biochemical and molecular studies of OC differentiation and function in a single species. We used this system to characterize the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the expression of proteins that bind to the serum response element (SRE) of the c-fos promoter. We found that OCLs and OCs displayed similar SRE-binding activities, including the serum response factor (SRF). This pattern is established in a time-dependent and cell-specific manner in response to long-term treatment of rabbit bone marrow by 1,25(OH)2D3. Thus, 1,25(OH)2D3 can modulate SRF and/or SRF-related protein. This finding may contribute to understanding the role of c-Fos in the regulation of OC differentiation.
A New Perspective on the Causal Influence of Soft Tissue Composition on DXA-Measured In Vivo Bone Mineral Density
Authors: Bolotin H. H.
Published: April 2006

An extensive series of quantitative simulation studies replicating ideal dual-energy X-ray absorptiometric (DXA) bone mineral density (BMD) measurements of typical and realistic in vivo lumbar vertebral and proximal femoral sites has been carried out to quantitatively assess the extent of inherent systematic inaccuracies in such measurements. The results for these bone sites indicate that BMD inaccuracies as high as 20% or more can be anticipated clinically, particularly in cases of osteopenic, osteoporotic, and elderly patients. It is found that the most important soft-tissue anthropometric determinants of the extent of bone site-specific systematic in vivo BMD inaccuracies reflected in DXA measurements are the ratio of the areal density of extraosseous fat to that of lean muscle tissue immediately surrounding the interrogated bone site and the specific yellow/red marrow mix within the scanned bone. As such, the present findings focus directly on the question of whether or not the strong, positive correlations and associations between soft tissue compositional parameters and DXA-measured in vivo BMD determined in a large number of previous clinical investigations are, in toto or in part, biologically causal. The present results are seen to be quantitatively and qualitatively in conformity with the many clinical studies that have found marked general decreases (increases) in measured BMD as body weight and/or body fat mass decreases (increases). It is concluded that the clinically observed correlations between DXA-measured BMD and these anthropometric parameters are artefacts of the systematic errors (inaccuracies) inherent in planar DXA methodology and are unlikely to be of biological genesis.
Mechanism of Mechanically Induced Intercellular Calcium Waves in Rabbit Articular Chondrocytes and in HIG-82 Synovial Cells
Authors: Grandolfo Micaela , Calabrese Alessandra , D'andrea Paola
Published: April 2006

Intercellular communication through gap junctions allows tissue coordination of cell metabolism and sensitivity to extracellular stimuli. Intercellular Ca2+ signaling was investigated with digital fluorescence video imaging in primary cultures of articular chondrocytes and in HIG-82 synovial cells. In both cell types, mechanical stimulation of a single cell induced a wave of increased Ca2+ that was communicated to surrounding cells. Intercellular Ca2+ spreading was inhibited by 18α-glycyrrhetinic acid, demonstrating the involvement of gap junctions in signal propagation. In the absence of extracellular Ca2+, mechanical stimulation induced communicated Ca2+ waves similar to controls; however, the number of HIG-82 cells recruited decreased significantly. Mechanical stress induced Ca2+ influx both in the stimulated chondrocyte and HIG-82 cell, but not in the adjacent cells, as assessed by the Mn2+ quenching technique. Treatment of cells with thapsigargin and with the phospholipase C (PLC) inhibitor U73122 blocked mechanically induced signal propagation. These results provide evidence that in chondrocytes and in HIG-82 synovial cells, mechanical stimulation activates PLC, thus leading to an increase of intracellular inositol 1,4,5-trisphosphate. The second messenger, by permeating gap junctions, stimulates intracellular Ca2+ release in neighboring cells. It is concluded that intercellular Ca2+ waves may provide a mechanism to coordinate tissue responses in joint physiology.
Calciotropic Hormones and Bone Markers in the Elderly
Authors: Gallagher J. Christopher , Kinyamu H. Karimi , Fowler Sarah E. , Dawson-Hughes Bess , Dalsky Gail P. , Sherman Sherry S.
Published: April 2006

There is a lack of substantial data on changes in calciotropic hormones and bone markers in elderly subjects living in North America. Parathyroid hormone (PTH), serum 25-hydroxyvitamin D (25(OH)D) and bone markers (serum osteocalcin and urine N-telopeptide), were measured in 735 Caucasian subjects (235 men and 500 women) aged 65–87 years. There was a significant increase in serum osteocalcin and urine N-telopeptide with age in men, and a significant increase in serum osteocalcin with age in women. Serum PTH and 25(OH)D showed no significant change with age in men or women. After adjusting for age, calcium intake, serum creatinine, season, and weight, mean serum PTH (p = 0.01), serum osteocalcin (p = 0.0001) and 24 h urine N-telopeptide (p = 0.0001) were higher in women than men, and mean serum 25(OH)D (p = 0.0001) and 24 h urine calcium (p = 0.0001) were higher in men than women. Serum PTH was correlated with serum osteocalcin in men and women, r = 0.24, r = 0.17, p < 0.001, but not with urine N-telopeptide. Serum PTH was inversely correlated with serum 25(OH)D (r = −0.25, r = −0.34, p < 0.001), and positively correlated with serum creatinine (r = 0.14, r = 0.17, p < 0.01) in men and women. The prevalence of serum 25(OH)D levels below 12 ng/ml was only 3.3% in females and 0.4% in men. Thus vitamin D deficiency was very uncommon in the U.S.A. compared with Europe. Although mean serum PTH was increased in the elderly, only 4–6% had PTH levels above the normal range. In summary, the increase in serum PTH in the elderly can be explained more by changes in vitamin D status than by declining renal function. These data also show significantly higher (p = 0.001) bone remodeling markers in women.
Bone Turnover Response to Changes in Calcium Intake Is Altered in Girls and Adult Women in Families with Histories of Osteoporosis
Authors: O'Brien Kimberly O. , Abrams Steven A. , Liang Lily K. , Ellis Kenneth J. , Gagel Robert F.
Published: April 2006

Heredity and environmental factors contribute to the development of osteoporosis. Because calcium is the major mineral in bone and adolescence is a key period in bone acquisition, we hypothesized that bone turnover would be less responsive to alterations in dietary calcium intake in both girls and adult women from families with histories of osteoporosis. To address this issue, we studied calcium kinetics in the maternal grandmother (age range 56–81 years), mother (age range 32–47 years), and granddaughter (age range 8–15 years) in 10 multigenerational families. In five families, the mother and/or grandmother had osteoporosis (bone mineral density ≥ 2 SD below the age-specific mean). To examine both active and passive calcium absorption, families consumed low- (279 ± 64 mg/day) and high- (1580 ± 385) calcium diets for 10 days prior to administration of oral (46Ca) and intravenous (42Ca) stable isotopes. Using repeated measures analysis of variance, fractional calcium absorption, true calcium absorption, bone calcium deposition, and the balance in bone calcium turnover were all significantly affected by diet (p < 0.01). Females from nonosteoporotic families had decreased bone calcium resorption with little change in bone calcium deposition during the high-calcium study. In contrast, girls and adult women from osteoporotic families had increased both bone calcium deposition and resorption during the high-calcium period, leading to a less positive balance in bone calcium turnover. A significant interaction between bone status and diet was found for bone calcium resorption (p < 0.05) and approached significance for bone calcium deposition (p < 0.07), effects which were independent of generation. We conclude that girls and women from osteoporotic families have a significantly altered bone turnover response to acute changes in calcium intake.