bluuebunny/crossref_metadata_2025 huggingface dataset:
Importance of bone resorption in the determination of bone density in women more than 10 years past the menopause
Authors: Dr. Prince Richard , Dick Ian , Devine Amanda , Kerr Deborah , Criddle R. Arthur , Price Roger , Webb Peter Garcia
Published: February 2010

There is general agreement that bone density falls with age and is higher in heavy people than light people. We have studied a variety of potential correlates of vertebral, ankle, and hip bone density to evaluate other potential influences on the skeleton. We recruited 196 healthy women who were more than 10 years past the menopause and collected a diet and activity record, a 24 h urine, and a fasting blood and urine specimen. These blood and urine samples were analyzed for factors related to calcium homeostasis. We then measured bone density at lumbar vertebrae 1–4 and the hip and the ankle bone density of the nondominant leg. Correlations between vertebral, hip, and ankle bone density and other measured variables were explored using the statistical package SPSS PC. At the vertebral site, in addition to correlations with age and body mass index (BMI), a negative correlation with a measure of bone resorption, the hydroxyproline creatinine ratio (OHPCR), was noted. At the ankle site, in addition to correlation with age, BMI, and OHPCR, a positive correlation with activity and a negative correlation with serum calcitriol were noted. At the hip site, as well as age, BMI, and OHPCR, significant correlations with GFR and dietary calcium intake were noted. These data suggest that even in women 10 years past the menopause bone resorption has a significant effect on bone density, that renal function may account for some of the variance in bone density at the hip, and that activity effects are more marked at sites of greater loading, namely the ankle.
Streaming potentials in healing, remodeling, and intact cortical bone
Authors: MacGinitie Laura A. , Wu D.D. , Cochran G.V.B.
Published: February 2010

Electrical fields have been implicated in accelerated bone healing and as a transduction mechanism for mechanically driven bone remodeling. Applied mechanical or electrical stimulation of bone remodeling suggests that this depends on the magnitude, frequency, and duration of the stimulus. The magnitude of endogenous electrical fields, manifest by streaming potentials (SPs) across canine cortical bone, were measured as a function of bending frequency in vivo and then in vitro at healing drill holes and at remodeling (ipsilateral) and normal, intact (contralateral) control sites in canine tibia. SP magnitudes normalized to periosteal strain were smaller for drill holes at 2 and 4 weeks postsurgery relative to either remodeling (P < 0.05 at 10 Hz) or normal intact (P < 0.001 at 10 Hz) controls both in vivo and in vitro. SPs of 12 week drill holes were similar to SPs of remodeling controls and tended to be smaller than SPs of normal intact controls. Mean SP normalized to bone impedance was approximately the same for all sites, suggesting that the smaller SPs during healing and remodeling relate to smaller bone impedance and/or larger porosity. SP as a function of bending frequency for normal sites was similar to that observed previously. SP versus frequency for drill holes and remodeling controls was more variable, probably because of variations in bone microstructure, and displayed a higher frequency content. The observed differences in SP magnitude and frequency response to loading associated with stages of healing indicate that endogenous electrical fields do indeed respond to the structural changes in healing and remodeling and are therefore capable of providing structural feedback information for the repair and remodeling process.
A 5-year longitudinal study of forearm bone mass in 307 postmenopausal women
Authors: Prof. Nordin B.E. Christopher , Cleghorn David B. , Chatterton Barry E. , Morris Howard A. , Need Allan G.
Published: February 2010

We measured forearm bone mineral content at the beginning and end of a 5 year period in 307 untreated postmenopausal volunteers. We also measured height, weight, and a number of biochemical variables in plasma and urine after an overnight fast. The initial mean age of the subjects was 59.0 years (range 39–72), and the mean years since menopause was 10.0 (range 1–37). The mean forearm BMC fell from 1034 ± 9.6 (SEM) to 982 ± 9.3 mg/cm (P < 0.001). The coefficient of correlation between the first and second measurements was 0.96. The mean rate of change was −1.0% per annum (with a 99% range of −4 to 1% per annum), which agreed well with previous estimates from cross-sectional data. There was a significant negative correlation between rate of change in bone mass and initial value (r = −0.23; P < 0.001), which was eliminated by expressing change as a percentage of initial bone mass. Of the other variables measured, the one that was most significantly related to the percentage change in bone mass was the urinary hydroxyproline/creatinine ratio (r = −0.35; P < 0.001), which we regard as a marker only. By stepwise regression, the only significant determinants of the rate of change in bone mass were body weight (positive, P < 0.001), years since menopause (positive, P < 0.005), urine calcium (negative, P < 0.01), and serum estrone (positive, P < 0.05). The rate of change in BMC was not significantly related to dietary calcium, plasma alkaline phosphatase, serum DHEAS, or any other variable measured at the outset or to serum PTH or serum osteocalcin measured at the conclusion.
Changes in markers of bone formation and resorption in a bed rest model of weightlessness
Authors: Lueken Sandra A. , Arnaud Sara B. , Dr. Taylor Arch K. , Baylink David J.
Published: February 2010

To study the mechanism of bone loss in physical unloading, we examined indices of bone formation and bone resorption in the serum and urine of eight healthy men during a 7 day −6° head-down tilt bed rest. Prompt increases in markers of resorption — pyridinoline (PD), deoxypyridinoline (DPD), and hydroxyproline (Hyp)/g creatinine—during the first few days of inactivity were paralleled by tartrate-resistant acid phosphatase (TRAP) with significant increases in all these markers by day 4 of bed rest. An index of formation, skeletal alkaline phosphatase (SALP), did not change during bed rest and showed a moderate 15% increase 1 week after reambulation. In contrast to SALP, serum osteocalcin (OC) began increasing the day preceding the increase in Hyp, remained elevated for the duration of the bed rest, and returned to pre-bed rest values within 5 days of reambulation. Similarly, DPD increased significantly at the onset of bed rest, remained elevated for the duration of bed rest, and returned to pre-bed rest levels upon reambulation. On the other hand, the other three indices of resorption, Hyp, PD, and TRAP, remained elevated for 2 weeks after reambulation. The most sensitive indices of the levels of physical activity proved to be the noncollagenous protein, OC, and the collagen crosslinker, DPD. The bed rest values of both these markers were significantly elevated compared to both the pre-bed rest values and the post-bed rest values. The sequence of changes in the circulating markers of bone metabolism indicated that increases in serum OC are the earliest responses of bone to head-down tilt bed rest.
Effects of high calcium concentration on the functions and interactions of osteoblastic cells and monocytes and on the formation of osteoclast-like cells
Authors: Sugimoto Toshitsugu , Kanatani Masanori , Kano Junichi , Kaji Hiroshi , Tsukamoto Tatsuo , Yamaguchi Toru , Fukase Masaaki , Chihara Kazuo
Published: February 2010

The present study was performed to clarify the role of high calcium concentration and the appearance of mononuclear cells at the resorptive site in bone remodeling. Our recent study revealed that the high concentration of extracellular calcium ([Ca2+]e) stimulated DNA synthesis in osteoblastic MC3T3-E1 cells not only directly but also indirectly via monocytes. Human monocyte-conditioned medium (CM) significantly stimulated DNA synthesis and inhibited alkaline phosphatase (ALP) activity. In contrast, when monocytes were cultured at high [Ca2+]e concentrations (more than 3 mM), CM from these monocytes significantly stimulated ALP activity in MC3T3-E1 cells. Such stimulatory effect of CM was not observed at a high magnesium concentration (Mg2+, 5 mM). Treatment of monocytes with the calcium ionophore A23187 did not affect the CM-induced effect on DNA synthesis and ALP activity in these cells. To determine the migration potency of MC3T3-E1 cells and monocytes toward the high [Ca2+]e, chemotaxis assay was performed. The increasing [Ca2+]e (more than 3 mM) induced a chemotactic response of MC3T3-E1 cells as well as monocytes, but the high concentration of Mg2+ (5 mM) did not induce it. On the other hand, treatment with high [Ca2+]e (more than 3 mM) or CM significantly inhibited the 1,25-(OH)2D3-induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC) from their precursors derived from mouse spleen cells. The present study indicated that an increase in [Ca2+]e stimulated DNA synthesis and ALP activity of osteoblasts via monocytes, induced chemotaxis of osteoblasts as well as monocytes, and inhibited the formation of TRAP-positive MNC, suggesting the importance of the high Ca2+ concentration and mononuclear cells at the resorptive site in bone remodeling.
Intranasal calcitonin suppresses increased bone resorption during short-term immobilization: A double-blind study of the effects of intranasal calcitonin on biochemical parameters of bone turnover
Authors: van der Wiel H.E. , Lips P. , Nauta J. , Kwakkel G. , Hazenberg G. , Netelenbos J.C. , van der Vijgh W.J.F.
Published: February 2010

Immobilization is associated with increased bone resorption and decreased bone formation. We evaluated in a double-blind trial the effect of intranasal administration of salmon calcitonin on biochemical parameters of bone turnover in 32 patients immobilized for a prolapsed intervertebral disk. Calcitonin in a dose of two times 200 IU/day partially inhibited the increase in the fasting 2 h urinary hydroxyproline/creatinine ratio (OHPr/Cr) and calcium/creatinine ratio (Ca/Cr). The increase in OHPr/Cr was 40% less in the calcitonin group compared to the placebo group (P = 0.01), and the increase in Ca/Cr was 80% less in the calcitonin group (P = 0.04). Calcitonin also partially inhibited the increase in serum cross-linked carboxyl-terminal telopeptide of collagen type I (P < 0.05). The decrease in serum 1,25-dihydroxyvitamin D after 10 days of immobilization was significantly less in the calcitonin-treated group than in the placebo group (14 versus 29%, respectively; P < 0.05). Intranasal calcitonin did not influence the pain scores as measured with a visual analog scale (VAS). The tolerability of the nasal calcitonin preparation was excellent. We conclude that nasal salmon calcitonin counteracts the early increase in bone resorption induced by immobilization.
Determinants of remission of paget's disease of bone
Authors: Patel S. , Stone M.D. , Coupland C. , Dr. Hosking D.J.
Published: February 2010

Bisphosphonates are a safe and effective treatment for Paget's disease of bone, but little information is available about the factors influencing the duration of remission so obtained. We assessed 60 patients with Paget's disease treated with disodium pamidronate (APD). The mean duration of remission was 9.5 months (range 3–25). The major influences were the initial pretreatment alkaline phosphatase (ALP; r = −4.6, p < 0.0001), minimum posttreatment ALP (r = −0.51, p < 0.0001), and the rate of response of bone turnover to the first dose of APD (r = 0.61, p < 0.0001). Multiple linear regression showed that the initial response to treatment was the most significant influence. Also, despite a minimum ALP within the normal range, the duration of remission varied considerably (4–25 months). This may be due to the difficulties in applying a population-based normal range to individuals.
Macrophage colony-stimulating factor release and receptor expression in bone cells
Authors: Dr. Weir Eleanor C. , Horowitz Mark C. , Baron Roland , Centrella Michael , Kacinski Barry M. , Insogna Karl L.
Published: February 2010

Colony-stimulating factors (CSF) may play a role in bone resorption. To examine whether osteoblasts secrete colony-stimulating activity (CSA) in response to parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), conditioned medium (CM) from ROS 17/2.8 cells and primary rat osteoblasts were examined for induction of clonal growth of cultured rat bone marrow cells. Untreated cells constitutively secreted CSA, which increased with PTH and PTHrP treatment. The colonies formed were principally comprised of macrophages, and preincubation of CM with antiserum to murine macrophage colony-stimulating factor (M-CSF) neutralized most of the CSA, suggesting that the osteoblast-derived CSA was predominantly due to M-CSF. PTHrP treatment upregulated steady-state M-CSF mRNA levels. To investigate a paracrine role for M-CSF in bone we examined bone tissue and cells for the M-CSF receptor c-fms using immunohistochemical techniques and demonstrated staining of mature osteoclasts both in situ and after isolation. We conclude that M-CSF is responsible for the majority of the CSA released by PTH- and PTHrP-treated rat osteoblasts. In addition we identified CSF-1 receptor expression in mature osteoclasts. These data suggest that M-CSF is a mediator of osteoblast-osteoclast interaction in PTH- and PTHrP-induced bone resorption.
Journal of Bone and Mineral Research
Authors: Veis Arthur
Published: February 2010

The bone, dentin, and cementum of the mature individual are comprised from a dense collagenous fiber network into which the carbonate-apatite mineral phase is deposited. It is hypothesized that a set of collagen-interactive acidic phosphoproteins are secreted by the osteoblasts, odontoblasts, and cementoblasts into the preformed collagenous matrix. These proteins then interact specifically with the collagen and nucleate apatite formation on and within the fibrils. These phosphoproteins may also regulate the morphology, rate of growth, and stability of the mineral phase crystals. The acidic matrix phosphoproteins may thus be considered as the crucial regulators of mineralization and tissue stability. In the dentin system, these regulatory proteins are synthesized, posttranslationally modified, and secreted in vesicles different from the collagen secretory vesicles. Mineralization occurs as the regulatory proteins are deposited on the preformed fibrils. This model requires testing in the bone system. In dentin, in the absence of tissue turnover, the resident phosphoproteins are degraded in situ over time, perhaps changing the properties of the tissue. Regulation of synthesis, secretory pathways and retention of integrity within the matrix are thus important areas for further investigation.
Journal of Bone and Mineral Research
Authors: Cann Christopher E.
Published: February 2010

The primary role of radiographic imaging in dental health has been the detection and localization of disease as a guide for surgical intervention. In this role, it appears to differ from the primary role of postcranial skeletal radiography, evaluation of the structural integrity of the bone. However, in both cases, the radiographic information ultimately is used in an attempt to restore or maintain function in the affected osseous structure. This structure-function relationship can be used as a framework within which the potential of new technologies developed for evaluation of the postcranial skeleton can be assessed relative to craniofacial imaging. Within this context, I treat first the use of newer imaging modalities, followed by methods to assess bone properties, and finally the coupling of these technologies with other analytic tools to obtain direct information about the structure-function relationship in the cranioskeleton.
CD44 expression in human bone: A novel marker of osteocytic differentiation
Authors: Hughes D.E. , Salter D.M. , Simpson R.
Published: February 2010

CD44 is a transmembrane glycoprotein with cell-cell and cell-matrix adhesion functions that is expressed by a wide variety of cell types and has a number of known biologic functions. Because of its ability to bind matrix macromolecules, such as fibronectin, collagen, and hyaluronate, we investigated the possibility that it is expressed by the cells of bone, the matrix receptors of which are largely unknown. Immunohistochemical study of a variety of sources of human bone was carried out using a panel of six well-characterized anti-CD44 monoclonal antibodies. Osteocytes strongly expressed CD44, whereas osteoblasts and lining cells were negative. Osteoclasts and periosteal cells also expressed CD44, although not as strongly as osteocytes. These patterns of staining were observed with all six antibodies. These results demonstrate that acquisition of CD44 immunoreactivity is a sensitive marker of osteocytic differentiation and raise the possibility that CD44 acts as a cell matrix receptor in bone.
Transcriptional activation of the human osteocalcin gene by basic fibroblast growth factor
Authors: Schedlich Lynette J. , Flanagan Judith L. , Crofts Linda A. , Gillies Susan A. , Goldberg Daniella , Morrison Nigel A. , Eisman John A.
Published: February 2010

Basic fibroblast growth factor (bFGF) has been detected in bone cells and stimulates osteoblast proliferation; however, its role in the regulation of bone metabolism remains speculative. We demonstrated that the human osteocalcin promoter is activated by bFGF when transfected into rat osteoblastic (ROS 17/2.8) cells. This effect is concentration dependent, with a twofold induction at 10 ng/ml detected after 20 h. The bFGF response is independent of both the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and retinoic acid activation of the osteocalcin promoter. To identify the promoter sequences through which bFGF exerts its effect, we tested a series of promoter deletion constructs for their response to bFGF. Deletion of the upstream region between −673 and −588 bp results in a significant loss of induction. Gel-shift analysis demonstrates that proteins present in ROS 17/2.8 nuclear extracts bind specifically to these sequences. This region alone was unable to confer the bFGF response on a minimal osteocalcin or an heterologous promoter. However, sequences between −678 and −476 bp, which also includes the vitamin D response element (VDRE), were able to confer bFGF inducibility on both a minimal osteocalcin and a heterologous promoter. These data suggest that induction of the human osteocalcin promoter by bFGF requires the interaction of more than one sequence element.
Role of chondroitin sulfate glycosaminoglycans in mineralizing osteoblast-like cells: Effects of hormonal manipulation
Authors: Slater M. , Patava J. , Mason R.S.
Published: February 2010

A monoclonal anti-chondroitin sulfate antibody (CS-56) that recognizes native chondroitin sulfate glycosaminoglycans (CSGAG) was used to quantify changes in CSGAG labeling levels in mineralizing human fetal osteoblast-like cell multilayers up to 40 days postconfluence. In control cultures, mean labeling of CSGAG increased in nonmineralized areas from around eight gold probes per μm2 (gpm) at 20 days to 26 gpm at 40 days. Labeling was markedly increased in the mineralized tissue, to 560 gpm at 30 days and 580 gpm at 40 days. In β-glycerophosphate-treated cultures, the mineralized areas were increased and appeared earlier (20 days) than in the control cultures. In these cultures, mean CSGASG labeling increased in nonmineralized areas from around 5 gpm at 20 days to 26 gpm at 30 days and was further increased in mineralized areas to 270 gpm at 20 days and 298 gpm at 30 days. Mineralization was not noted in cultures treated with 10−8 M 1,25-dihydroxyvitamin D, and CSGAG labeling remained low (<5 gpm) during the study period. These results indicate that an increase in immunoreactive CSGAG is associated with mineralization in this culture system. One possible interpretation of these findings is that proteoglycan molecules or at least their CSGAG side chains may be involved in the mineralization process.
Characterization of the stromal osteogenic cell line MN7: mRNA steady-state level of selected osteogenic markers depends on cell density and is influenced by 17β-estradiol
Authors: Mathieu E. , Dr. Merregaert J.
Published: February 2010

The steady-state mRNA levels of different osteogenic markers and their modulation by 17β-estradiol in the murine osteogenic cell line MN7 during proliferation and differentiation in vitro were examined. mRNA of collagen type I, osteopontin, bone morphogenetic protein 2, plasminogen activator inhibitor 1, alkaline phosphatase, and osteocalcin were isolated from MN7 cultures grown for 7, 11, 14, and 17 days. Northern blot analysis revealed steady-state transcript levels depending on MN7 cell density. The order of appearance of Col I, OP, ALP, and OC resembled the pattern of gene expression observed during osteoblast maturation in vitro. Furthermore, PAI-1 steady-state transcript levels peaked during subconfluence (day 11) but BMP-2 RNA levels reached their maximum after the culture had become confluent. 17β-Estradiol showed a dose-dependent stimulation of the different osteoblast-related transcripts present in a subconfluent MN7 culture at the time of analysis. Furthermore, the effects of 17β-estradiol (17βE2) at different time points of MN7 growth varied according to cell density. 17βE2 added to subconfluent MN7 cultures modulated the transcript level in a negative way, but RNA levels of the investigated osteogenic markers in confluent cultures were stimulated with 100 nM 17β-estradiol. No effect of 17β-estradiol on proliferation was detected. The present studies have revealed differential osteoblast gene expression related to MN7 cell proliferation and differentiation in vitro and emphasize the importance of 17βE2 in the regulation of growth of this preosteoblastic cell line in vitro.
Temporal changes in matrix protein synthesis and mRNA expression during mineralized tissue formation by adult rat bone marrow cells in culture
Authors: Yao Kam-Ling , Todescan Reynaldo , Dr. Sodek Jaro
Published: February 2010

To characterize the bone-like tissue produced by rat bone marrow cells (RBMC) from young adult femurs, the synthesis of bone proteins and the expression of their mRNA were studied in vitro. RBMC plated at a density of 5 × 103 cells/cm2 and grown in the presence of 10−8 M dexamethasone (Dex) and 10 mM β-glycerophosphate (β-GP) produced mineralized bone nodules, which were first evident at day 3 and increased markedly to day 13. However, in the absence of dexamethasone, few mineralized nodules were observed. The formation of mineralized nodules was reflected by the uptake of 45Ca, which also increased markedly to day 13. Analysis of bone protein expression by Northern and slot-blot hybridizations revealed an increase in mRNA levels of collagen type I (Col I), osteonectin/SPARC (ON), alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OC) during the formation of mineralized nodules. Whereas the Col I, ON, ALP, and OPN mRNAs were expressed before the formation of mineralized nodules was evident and were also expressed at various levels in the absence of Dex, the expression of BSP and OC mRNA was induced in the bone-forming cultures. The expression of BSP mRNA was correlated temporally with bone tissue formation, reaching maximal levels on day 16. In contrast, OC mRNA was expressed later and, following induction, increased over the 28 day culture period. Production of matrix proteins during the rapid formation of the bone tissue appeared to reflect the levels of the respective mRNAs. However, whereas some of the collagen and almost all of the SPARC were secreted into the culture medium, virtually all of the OPN and most of the BSP were extracted from the mineralized tissue matrix with EDTA. Some OPN and BSP were present in the medium, especially early in the culture, and a significant amount of BSP was also found associated with the collagenous tissue matrix. These studies point to the importance of Col I, ALP, OPN, and BSP, but not ON or OC, in the initial formation of bone tissue.
Upstream regulatory elements necessary for expression of the rat COL1A1 promoter in transgenic mice
Authors: Bogdanovic Zoran , Bedalov Antonio , Krebsbach Paul H. , Pavlin Dubravko , Woody Charles O. , Clark Stephen H. , Thomas Huw F. , Rowe David W. , Kream Barbara E. , Lichtler Alexander C.
Published: February 2010

The activity of fusion genes containing fragments of the COL1A1 promoter was measured in tissues from 6- to 8-day-old transgenic mice. ColCAT3.6 contains approximately 3.6 kb (-3521 to 115 bp) of the rat COL1A1 gene, the chloramphenicol acetyltransferase (CAT) reporter gene, and the SV40 splice and polyadenylation sequences. ColCAT2.3 and ColCAT1.7 are deletion constructs that contain 2296 and 1667 bp of COL1A1 upstream from the RNA start site, respectively. For each transgene, up to six lines of mice were characterized. Both ColCAT3.6 and ColCAT2.3 had similar activity in bone and tooth; ColCAT1.7 was inactive. In transgenic calvariae, levels of transgene mRNA paralleled levels of CAT activity. In tendon, the activity of ColCAT2.3 was 3- to 4-fold lower than that of ColCAT3.6, and the activity ColCAT1.7 was 16-fold lower than that of ColCAT2.3. There was little activity of the ColCAT constructs in liver and brain. These data show that DNA sequnces between −2.3 and −1.7 kb are required for COL1A1 promoter expression in bone and tooth; sequences that control expression in tendon are distributed between −3.5 and −1.7 kb of the promoter, with sequences downstream of −1.7 kb still capable of directing expression to this tissue. The cis elements that govern basal expression of COL1A1 in transgenic calvariae appear to be different from those required for optimal expression of the COL1A1 promoter in stably transfected osteoblastic cells.
Influence of early age at menopause on vertebral bone mass
Authors: Pouillès J.M. , Trémollières F. , Bonneu M. , Dr. Ribot C.
Published: February 2010

Menopause leads to rapid bone loss, mainly as a result of estrogen deficiency superimposed on the age-related linear bone loss. The influence of age at menopause on bone loss is unclear, although early menopause is widely considered a risk factor for osteoporosis. Vertebral bone mineral density (BMD) was measured in 1667 women divided into five groups according to hormonal status and age at menopause. Menopausal status was an independent predictor of BMD in a multiregression analysis, along with current age, years since menopause (YSM), weight, and height. For the same chronologic age (55 years), women with early menopause had a 15% lower BMD and a higher YSM than women whose menopause occurred later (“normal” menopause). After adjusting for the interval since menopause, postmenopausal women with early menopause were found to have lower vertebral BMD than postmenopausal women with normal menopause. Finally, after the age of 60,66% of the women with early menopause had a BMD that was below the fracture threshold compared to 18% of the women with normal menopause. The results of this cross-sectional study suggest that early menopause is associated with a quantitatively higher bone loss than in women with menopause of later onset and thus constitutes a risk factor for osteoporosis.
Characterization, regulation, and function of specific cell membrane receptors for insulin-like growth factor I on bone endothelial cells
Authors: Fiorelli Gianna , Orlando Claudio , Benvenuti Susanna , Franceschelli Francesco , Bianchi Sandro , Pioli Piero , Tanini Annalisa , Serio Mario , Bartucci Francesco , Dr. Brandi Maria Luisa
Published: February 2010

It is now widely accepted that insulin-like growth factor-I (IGF-I) has a local regulatory role in bone remodeling. IGF-I has also been demonstrated to regulate proliferation of bone-derived endothelial cells. Such studies suggest a role of IGF-I in skeletal angiogenesis. Using BBE cells, a bovine bone endothelial cell line, we characterized the kinetics and chemical properties of IGF-I receptors and examined the effect of IGF-I on bone endothelium migration. Two classes of binding sites with high affinity for IGF-I were detected by binding experiments on bone endothelial cells. Both competition analyses and cross-linking studies revealed the presence of type I IGF receptor in bone endothelial cells. Moreover, these cells produced and released authentic IGF-I into the medium, as evidenced by radioimmunoassay analyses of gel-filtered conditioned media. Both IGF-I binding capacity and release decreased either with increases in cell number or after treatment with 17β-estradiol (17βE2) and parathyroid hormone (PTH). Both hormones also inhibited chemotactic responses of bone endothelial cells to IGF-I. Taken together, these results strongly suggest that IGF-I, a growth factor that promotes the proliferation of various bone cell types, also induces growth and chemotactic responses in bone endothelium acting through the type I IGF receptor. This may be part of a generalized response of bone cells to IGF-I that facilitates cell migration.
Cloning and sequence analysis of bovine bone sialoprotein cDNA: Conservation of acidic domains, tyrosine sulfation consensus repeats, and RGD cell attachment domain
Authors: Chenu C. , Ibaraki K. , Robey P. Gehron , Delmas P.D. , Young M.F.
Published: February 2010

We isolated and sequenced a cDNA encoding bovine bone sialoprotein (BSP) using a bovine cDNA library made from mRNA isolated from bone-derived cell cultures and ligated to a phage λgt11. One of the cDNA clones isolated from this library had a 1800 base pair long insert and was found to contain the entire protein-encoding region. The deduced protein sequence revealed a 310 amino acid protein containing a signal peptide sequence of 16 hydrophobic amino acids. The protein sequence shows remarkable conservation with previously published human and rat sequences (more than 80% similarity for both species). The potential functional domains of BSP, including three acid amino acid-rich sequences, tyrosine sulfation consensus repeats, and the RGD cell binding sequence, are all present in the bovine sequence. Northern analysis of RNA from different bovine tissues indicated the presence of BSP message in bone but not in other nonmineralized tissues, confirming that bone is the major site of BSP message production.
Osteoclast synthesis and secretion and activation of latent transforming growth factor β
Authors: Oursler Merry Jo
Published: February 2010

The coupling of bone resorption and formation suggests that autocrine and paracrine factors are produced and released within the local bone environment. Since osteoblasts secrete latent transforming growth factor β (TGF-β), and osteoclasts can activate this latent complex, one candidate paracrine coupling factor is TGF-β. To examine whether TGF-β is also a potential osteoclast-derived autocrine and paracrine factor, highly purified avian osteoclasts were examined for synthesis of TGF-β and the mechanism by which osteoclasts activate the latent TGF-β complex was investigated. TGF-β protein production was measured within 4 h of culture in osteoclast-conditioned media using growth factor inhibition of CCL-64 cells and verified by blocking effects with anti-TGF-β antibodies. Synthesis of TGF-β was confirmed by northern blotting and metabolic labeling. Northern blots of total RNA revealed that osteoclasts expressed the mRNA for TGF-β2, β3, and β4. Biosynthetic studies suggest that TGF-β2 was the principle form secreted in culture. Nearly all the TGF-β that was secreted had been activated by the osteoclasts. When presented with exogenous latent TGF-β, osteoclasts activated latent TGF-β from a variety of sources. Furthermore, osteoclast-conditioned media retained the ability to activate latent TGF-β during cell-free incubations at 37°C. Osteoclast-mediated activation was inhibited by proteinase inhibitors, weak base treatment of the cells, or disruption of the cytoskeletal network. These data suggest that osteoclasts may secrete proteinases into the extracellular milieu to activate latent TGF-β and that TGF-β may be an autocrine factor involved in regulating osteoclast activity.
Phorbol myristate acetate stimulates osteoclast formation in 1α, 25-dihydroxyvitamin D3-primed mouse embryonic calvarial cells by a Prostaglandin-dependent mechanism
Authors: Amano Shigeru , Hanazawa Shigemasa , Kawata Yasuhiro , Nakada Yuh , Miyata Yuhko , Kitano Shigeo
Published: February 2010

Our previous study provided a novel assay system utilizing devitalized bone slices for study of the differentiation of osteoclast progenitors into preosteoclasts and mature osteoclasts among calvarial cells of mouse embryos. Using this assay system, we examined the effect of phorbol myristate acetate (PMA) on osteoclast formation as assessed by the appearance of tartrate-resistant acid phosphatase (TRAP)-positive cells and bone resorption lacunae. PMA alone was directly unable to induce the appearance of TRAP-positive cells and bone resorption lacunae of calvarial bone cells of mouse embryos. However, PMA markedly stimulated increases in the number of TRAP-positive cells and area of the resorption lacunae of the calvarial cells when the bone cells were primed by 1α,25-(OH)2D3. This stimulatory effect of PMA was dose dependent. H-7, having relatively high affinity for protein kinase C, strongly inhibited in a dose-dependent fashion the stimulatory effect of PMA on the bone resorption of the hormone-primed calvarial cells. We also examined the involvement of prostaglandin in this stimulatory effect of PMA. Indomethacin, a cyclooxygenase inhibitor, markedly abolished the stimulatory effect of PMA on the bone resorption of the calvarial cells. PMA stimulated prostaglandin E2 (PGE2) production by the calvarial cells primed with 1α, 25-(OH)2D3 in a dose-dependent fashion. However, the PMA stimulation of the PGE2 production was significantly inhibited by H-7 and also by indomethacin. Furthermore, we observed that the addition of PGE2 to the calvarial cells primed with 1α, 25-(OH)2D3 for 1 or 3 days resulted in an increased number of TRAP-positive cells and increased bone resorption. This stimulatory effect of the exogenous PGE2 was also observed in the presence of indomethacin. These results strongly suggest that PMA stimulates osteoclast formation in 1α, 25-(OH)2D3-primed calvarial cells by a prostaglandin-dependent mechanism.
Metabolic bone disease in black teenagers with genu valgum or varum without radiologic rickets: A bone histomorphometric study
Authors: Dr. Schnitzler Christine M. , Pettifor John M. , Patel Deepak , Mesquita Julia M. , Moodley Gopal P. , Zachen Dianne
Published: February 2010

Calcium deficiency in black (African) children can cause rickets and osteomalacia with severe limb deformities. It is not known whether black teenagers with genu valgum or varum but without radiologic rickets suffer from a related disorder. To examine this question we studied 26 such patients by iliac crest bone biopsy and serum and urine biochemistry: 12 patients (46%) had osteopenia with normal or low bone turnover, 5 (19%) mildly increased bone turnover, 4 (15%) histologic hyperparathyroidism, 2 (8%) preosteomalacia, and 3 (12%) osteomalacia (with features of hyperparathyroidism). Radiographs did not reflect the severity of the bone disease. Serum calcium levels correlated inversely with eroded mineralized surface (p < 0.001), osteoid surface (p < 0.01), osteoid thickness (p < 0.001), mineralization lag time (p < 0.001), and 1,25-(OH)2 vitamin D (p < 0.005), and 1,25-(OH)2 vitamin D correlated positively with osteoid surface (p < 0.05), osteoid thickness (p < 0.05), osteoid volume (p < 0.01), eroded surface (p < 0.05), and eroded mineralized surface (p < 0.0005). Tubular reabsorption of phosphate and 25-OH vitamin D levels were normal, and 1,25-(OH)2 vitamin D levels were normal to high. This suggests that calcium deficiency may have caused the increase in bone turnover and the mineralization defects. The most severe osteomalacia was found in males aged 16–19 years. We cannot explain the cause of the osteopenia. We conclude that all patients had bone disease.
Specific activity of skeletal alkaline phosphatase in human osteoblast-line cells regulated by phosphate, phosphate esters, and phosphate analogs and release of alkaline phosphatase activity inversely regulated by calcium
Authors: Farley John R. , Hall Susan L. , Tanner Michael A. , Wergedal Jon E.
Published: February 2010

We assessed the significance of Ca and phosphate (Pi) as determinants of (1) the amount of skeletal alkaline phosphatase (ALP) activity in SaOS-2 (human osteosarcoma) cells and normal human bone cells, and (2) the release of ALP activity from the cells into the culture medium. After 24 h in serum-free BGJb medium containing 0.25–2 mM Pi, the specific activity of ALP in SaOS-2 cells was proportional to Pi concentration (r = 0.99, p < 0.001). The Pi-dependent increase in ALP activity was time dependent (evident within 6 h) and could not be attributed to decreased ALP release, since Pi also increased the amount of ALP activity released (r = 0.99, p < 0.001). Parallel studies with Ca (0.25–2.0 mM) showed that the amount of ALP activity released from SaOS-2 cells was inversely proportional to the concentration of Ca (r = −0.85, p < 0.01). This effect was rapid (i.e., observed within 1 h) and could not be attributed to a decrease in the amount of ALP activity in the cells. Phase distribution studies showed that the effect of low Ca to increase ALP release reflected increases in the release of both hydrophilic ALP (i.e., anchorless ALP, released by phosphatidylinositol-glycanase activity) and hydrophobic ALP (i.e., phosphatidylinositol-glycan–anchored ALP, released by membrane vesicle formation). The range of Ca-dependent changes in ALP-specific activity was much smaller than the range of Pi-dependent changes. The observed correlation between skeletal ALP-specific activity and Pi was not unique to osteosarcoma cells or to Pi. Similar effects were seen in normal human bone cells in response to Pi (r = 0.99, p < 0.001) and in SaOS-2 cells in response to a variety of Pi esters and analogs (e.g., β-glycero-Pi and molybdate). Further studies indicated that the effects of phosphoryl compounds on ALP-specific activity could not be correlated with effects on ALP reaction kinetics, cell proliferation, or acid phosphatase activity and that the β-glycero-Pi-dependent increase in ALP activity was blocked by cycloheximide but not actinomycin D. Together these data suggest that the function of skeletal ALP may be regulated by Pi and that Ca may be involved in ALP release.
Effect of cyclosporin A on bone mineral metabolism in experimental diabetes mellitus in the rat
Authors: Epstein Sol , Takizawa M. , Stein B. , Katz I.A. , Joffe I.I. , Romero D.F. , Liang X.G. , Li M. , Ke H.Z. , Jee W.S.S. , Jacobs T.W. , Berlin J.
Published: February 2010

Cyclosporin A (CsA) is widely used in diabetic transplant patients and early type I diabetes mellitus. Diabetes produces a low-turnover osteopenia, and CsA conversely induces high-turnover osteopenia in rats. We investigated whether CsA would exacerbate diabetic osteopenia. Four groups of 10-week-old male Sprague-Dawley rats (n = 11/group) were studied: On day −6, groups A and C received saline and groups B and D received intravenous streptozotocin (55 mg/kg) to induce diabetes. From day 0, groups A and B received CsA vehicle and C and D received CsA (15 mg/kg) by daily gavage. Rats were bled on days −6, 0, 11, and 22 for serum bone gla protein (BGP), 1-,25-(OH)2D, PTH, blood ionized Ca, and blood glucose determinations. Double tetracycline labeling was performed on days 9 and 20 for bone histomorphometry. After sacrifice on day 22, histomorphometric analysis was performed. Serum BGP, 1,25-(OH)2D, and PTH levels were significantly decreased in the diabetic alone (B) and diabetic plus CsA (D) groups and significantly increased in the CsA alone (group C). CsA alone (group C) induced cancellous bone loss by stimulated bone resorption. Cancellous bone loss in the diabetic alone rats (group B) was caused primarily by inhibited bone formation. No differences were found in cancellous bone mass, formation, or resorption parameters between diabetic alone (group B) and CsA-treated diabetic rats (group D). Neither CsA alone (group C) nor diabetic alone (group B) nor their combination affected cortical bone mass. CsA alone (group C) stimulated periosteal bone formation and endocortical bone resorption and inhibited endocortical formation, and diabetic alone (group B) inhibited both periosteal and endocortical bone formation. No parameters of tibial diaphyses in CsA-treated diabetic rats (group D) were different from diabetic alone. Thus the addition of CsA to the diabetic treated rats (group D) could not stimulate remodeling and appeared not to worsen significantly some of the alterations in bone formation and resorption. Possible explanations for this may be that CsA in vivo requires adequate levels of PTH, 1,25-(OH)2D, insulin, and perhaps growth factors to stimulate remodeling. The use of CsA in type I diabetic patients or in organ transplant recipients who remain diabetic after transplantation may in the short term not aggravate existing osteopenia based on these findings.
Murine osteoclasts and spleen cell polykaryons are distinguished by mRNA phenotyping
Authors: Tong Hong-Sheng , Sakai Dennis D. , Sims Stephen M. , Dixon Jeffrey S. , Yamin Moshe , Goldring Steven R. , Snead Malcolm L. , Minkin Cedric
Published: February 2010

To probe osteoclast gene expression, we combined the techniques of cell microisolation and RT-PCR to develop a novel and sensitive method for the isolation and mRNA phenotyping of small numbers of authentic osteoclasts and spleen cell polykaryons. Using this method we report (1) direct evidence for the presence of calcitonin receptor mRNA in osteoclasts, (2) confirmation of the recent finding of osteopontin mRNA in osteoclasts, and (3) demonstration that the specific expression of mRNA for tartrate-resistant acid phosphatase, carbonic anhydrase II, calcitonin receptor, and osteopontin enable one to distinguish the osteoclast from the morphologically similar and developmentally related spleen cell polykaryon. We also show that mRNA associated with the osteoblast phenotype, such as alkaline phosphatase, osteocalcin, and type I collagen, are absent in osteoclasts. This is the first report in which such an approach has been used successfully to distinguish the mRNA expression pattern of an authentic osteoclast from a macrophage polykaryon, and as such it should provide an important new tool for evaluating the results of various cell culture model systems designed to examine the origin and ontogeny of osteoclasts. Our results also indicate that these procedures can be used as an alternative to in situ hybridization methods for the cell-specific localization of specific mRNA in a mixed cell preparation and for colocalization of multiple mRNA species to a single cell type.
Production and release of matrix vesicles in the cell processes of TPA-treated human osteoblast-like cells
Authors: Ringbom-Anderson Tove , Jäntti Jussi , Åkerman Karl E.O.
Published: February 2010

At the onset of the mineralization of bone, small membranous matrix vesicles are often observed. The information available on the production and release of these vesicles is limited. When treated with 10–20 nM of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), the human osteosarcoma cell line U-2 OS developed long cytoplasmic processes connecting adjacent cells. SEM and TEM show that TPA triggers a production and release of matrix vesicle-like membrane vesicles, mainly from the cellular processes. Tetracycline HC1 was used to label intracellular bound calcium. The tetracycline HC1 label was primarily localized to the end-feet of the cytoplasmic processes, indicating that these contain high concentrations of Ca2+, and to endoplasmic reticulum-like structures in the cell bodies. Together with our previous demonstration of the release of alkaline phosphatase-containing vesicles into the culture medium (Ringbom-Anderson T, Åkerman KEO 1992 Calcif Tissue Int 50:533–540), the results presented here indicate that TPA induces a rapid induction of the primary steps of mineralization in U-2 OS osteosarcoma.
Improved assessment of lumbar vertebral body strength using supine lateral dual-energy X-ray absorptiometry
Authors: Myers Barry S. , Arbogast Kristy B. , Lobaugh Bruce , Harper Kristine D. , Richardson William J. , Drezner Marc K.
Published: February 2010

Clinical and biomechanical investigations indicate that assessment of vertebral body bone mineral density (BMD) by anteroposterior dual-energy x-ray absorptiometry (DXA) is a useful index of vertebral body strength and fracture risk in osteoporosis. However, inclusion of non-force-bearing and small-force-bearing mineralized structures, such as the posterior elements and aortic calcifications, in the measurement of anterior BMD obscures the assessment of vertebral body mass by this technique. Indeed, such interference is particularly severe in the presence of posterior element degeneration or previous spinal surgery. Recent anatomic studies illustrate that the lateral view provides unobstructed visualization of the L3, L4, and possibly L2 vertebral bodies, suggesting that supine lateral BMD may more accurately assess vertebral body fracture risk. We evaluated this hypothesis in a blinded study using human cadaver spines to compare the value of supine lateral and anteroposterior BMD in assessing vertebral body fracture force, average compressive stress, maximum stored strain energy, and strain at failure. Both measures of BMD significantly correlate with these biomechanical measures. However, statistical comparison of the methods using multiple and stepwise regression reveals that supine lateral BMD provides a better assessment of the vertebral body fracture properties than anteroposterior BMD. The enhanced predictive value of supine lateral BMD occurs because of the variable contribution of posterior element mineral to the anteroposterior BMD measurement. Evaluation to test the utility of supine lateral BMD for the assessment of fracture risk and a fracture threshold in patients with osteoporosis is therefore recommended.
Hereditary hyperphosphatasia: 20 year follow-up and response to disodium etidronate
Authors: Singer Frank , Siris Ethel , Shane Elizabeth , Dempster David , Lindsay Robert , Parisien May
Published: February 2010

We provide a 20 year follow-up of a family with three siblings affected by hereditary hyperphosphatasia (HH). An iliac crest bone biopsy was performed on one of the siblings following double-tetracycline labeling, with results reported quantitatively in a standard histomorphometric format. Biochemical parameters of disease activity were monitored in the patient before and after treatment with oral etidronate disodium, 20 mg/kg/day taken for 5 weeks. Biochemical evidence of intense disease activity continued 20 years after the initial diagnosis of HH in the sibling studied. His bone biopsy specimen also revealed extremely high bone turnover but low cancellous bone volume and osteoclasts unlike those found in Paget's disease. Treatment with etidronate disodium resulted in a temporary 40% reduction in serum alkaline phosphatase and 24 h urine hydroxyproline excretion, with reduction in serum osteocalcin from two times the upper limit of normal to a subnormal level. We conclude that disease activity in HH can continue unabated for two decades. Our bone biopsy finding of low cancellous bone volume, the consistent lack of pagetic-looking osteoclasts in our and other studies, plus the clinical features of HH (childhood onset and extremely diffuse disease with gross skeletal deformation) serve to distinguish HH from Paget's disease. Bisphosphonates may be of value in treating HH.