bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo inhibition of localized bone resorption by human recombinant interleukin-1 receptor antagonist
Authors: Chole Richard A. , Faddis Brian T. , Tinling Steven P.
Published: February 2010

Interleukin-1 (IL-1) has been implicated as a primary mediator of bone remodeling; it is a powerful activator of bone resorption both in vivo and in vitro. However, there is no direct evidence that IL-1 plays a role in physiological bone modeling or remodeling. Interleukin-1 receptor antagonist (IL-1ra) is a member of the IL-1 family, which bind to IL-1 receptors and blocks the action of IL-1α and IL-1β. We have previously shown that IL-1ra blocks IL-1—induced bone resorption in vitro. Evidence is reported here that human recombinant IL-1ra (hrIL-1ra) inhibits strain-induced modeling in the gerbil auditory bulla while having no significant effect on apposition rate. Pressurization of the auditory bulla to 10 mm Hg above atmospheric pressure increased osteoclast surface from 3.62 to 19.14%. Infusion of hrIL-1ra during pressurization resulted in significant inhibition of osteoclast surface on the pressurized side. These findings suggest that IL-1 is a physiological mediator of bone modeling and that hrIL-1ra inhibits resorption but not apposition in the auditory bulla model.
Dexamethasone alters the subpopulation make-up of rat bone marrow stromal cell cultures
Authors: Herbertson Alexandra , Dr. Aubin Jane E.
Published: February 2010

Bone marrow stromal cells comprise a heterogeneous population including fibroblastic, adipocytic, hemopoietic, and osteogenic cells. Although the conditions under which different lineages are regulated have not been fully elucidated, dexamethasone clearly stimulates osteogenic expression in stromal cultures. The purpose of this study was to begin to elucidate and quantify some of the subpopulations present when rat bone marrow stromal cells are grown with or without dexamethasone under conditions favoring bone formation. Bone marrow stromal cells from young adult rats were cultured with ascorbic acid, β-glycerophosphate, and with or without dexamethasone for various periods of time. Culture dishes were then analyzed for cell counts, or stained with either histochemical or immunohistochemical stains, and colony types were quantitated, or cells were processed for flow cytometry. Dexamethasone significantly increased the number of alkaline phosphatase (AP) positive colonies, von Kossa positive bone nodules, α-naphthylbutyrate esterase positive colonies, and ED2 positive (macrophage) colonies. The number of adipocytic foci was largely unaffected in these experiments. Flow cytometry confirmed colony counts and showed stimulation by dexamethasone of AP positive cells and macrophages, and in addition, the reduction of hemopoietic cells expressing leukocyte common antigen. These data show conclusively that when rat bone marrow stromal populations are grown under conditions stimulating osteoprogenitor differentiation and bone formation, the stromal subpopulation make-up, including expression of hemopoietic lineages, is markedly altered.
Use of an in vivo model to determine the effects of interleukin-1 on cells at different stages in the osteoclast lineage
Authors: Uy Harry L. , Dallas Mark , Calland John W. , Boyce Brendan F. , Mundy Gregory R. , DR. Roodman David G.
Published: February 2010

In vitro model systems have been used extensively to study factors that affect osteoclast formation and to identify osteoclast precursors. However, in vitro systems do not examine the entire process of osteoclast differentiation simultaneously and lack accessory cells normally present in vivo. Additionally, the role that metabolism of the factor may play on its osteotropic activity in vivo is not addressed by these culture systems. Therefore, we have developed an in vivo model that permits us to examine simultaneously the effects of osteotropic factors on three distinct stages of osteoclast differentiation: (1) multipotent osteoclast precursors, the granulocyte-macrophage colony-forming unit (CFU-GM); (2) more differentiated marrow mononuclear osteoclast precursors; and (3) mature osteoclasts already present on bone surfaces. In the current study, we used interleukin-1 (IL-1) as a prototypic osteotropic factor to test the utility of this system to delineate the cellular mechanisms responsible for enhanced osteoclast activity stimulated by this cytokine. IL-1 induced hypercalcemia and enhanced the growth and differentiation of CFU-GM, increased the number of more committed mononuclear osteoclast precursors, and stimulated mature osteoclasts to resorb bone. These data demonstrate that this simple in vivo model permits the easy delineation of the stages of osteoclast development, in which osteotropic factors act to enhance bone turnover, and may be useful in understanding the mechanism of action of antiresorptive agents.
Bone resorption at the femoral neck is dependent on local factors in nonosteoporotic late postmenopausal women: An in vitro-in vivo study
Authors: Cohen-Solal M.E. , Graulet A.M. , Gueris J. , Denne M.A. , Bergot C. , Morieux C. , Sedel L. , Kuntz D. , Dr. de Vernejoul M.C.
Published: February 2010

Local mediators of bone resorption may be involved in bone loss in recently postmenopausal women and in osteoporosis. In the present study, we investigated the production of cytokines and the formation of osteoclast-like cells in marrow cultures from 16 late postmenopausal nonosteoporotic women (mean age: 66 ± 8 years; time after menopause: 15 ± 8 years) undergoing hip replacement for arthrosis. Marrow adherent mononuclear cells (MMNC) isolated from femoral diaphysis marrow were cultured for 10 days in the absence or in the presence of 1,25(OH)2D3. In vivo bone resorption was concomitantly assessed by histomorphometry on femoral neck bone sections. The number of TRAP+ multinucleated cells obtained after 10 days in MMNC cultured in the presence of 1,25(OH)2D3 correlated with the number of osteoclasts measured on the bone femoral neck biopsies (r = 0.65, p < 0.01), suggesting that the formation of multinucleated cells in vitro could reflect the osteoclast differentiation in vivo. Furthermore, the number of osteoclasts was related to the eroded volume and the trabecular separation of the femoral neck bone biopsies. Finally, the release of interleukin-1 (IL-1), IL-6, and TNF-α by cultures of peripheral blood mononuclear cells (PBMC) and MMNC was measured by radioimmunoassay. The cytokine levels of basal and 1,25(OH)2D3-treated MMNC decreased from days 2 to 5 and then reached a plateau to day 10. The number of TRAP+ multinucleated cells obtained after 10 days in MMNC cultures correlated with the basal IL-6 release in the same cultures determined at day 2 (r = 0.55, p < 0.01). IL-1 and IL-6 basal levels in PBMC and MMNC at day 2 were correlated (r = 0.53, r = 0.64, respectively; p < 0.05), showing that cytokine production in peripheral blood reflects the local production in the marrow. Our data indicate that, in late postmenopausal women, local factors, and in particular IL-6, might be implicated in the bone-resorbing process, and that osteoclast activity might be responsible for micro-structural changes in the femoral neck.
Comparison of speed of sound ultrasound with single photon absorptiometry for determining fracture odds ratios
Authors: Stegman Mary Ruth , Heaney Robert P. , Recker Robert R.
Published: February 2010

The purpose of this study was to compare the ability of ultrasound velocity measurement with that of single photon absorptiometry to determine the odds of history of fracture since age 40, for a population-based study. To do this we computed odds ratios and 95% confidence intervals for 809 women and 502 men, aged 50 years and older, who are participants in the prospective phase of the Saunders County Bone Quality Study. These participants received both the ultrasound and single photon absorptiometry bone measurements at the initiation of the study. In addition, a history of all fractures that had occurred to participants since age 40 was obtained. The two bone assessment methods were compared by examining the magnitude of the odds ratios, to determine which produces the highest estimate of the probability of odds of fracture, and by examining widths of the respective confidence intervals to show which estimate of odds ratio is the most precise. Ultrasound velocity estimates a higher probability of odds of both low-trauma fractures and all fractures than distal radius and ulna bone mineral content, but lower than bone mineral density at the same sites for both women and men. However, the ultrasound measure is more precise than bone mineral density, but less precise than bone mineral content. We conclude that ultrasound velocity is as good as single photon absorptiometry in estimating odds of fracture.
Effect of 1,25-dihydroxyvitamin D3 treatment on bone formation by transplanted cells from normal and X-linked hypophosphatemic mice
Authors: Ecarot B. , Glorieux F.H. , Desbarats M. , Travers R. , Labelle L.
Published: February 2010

Bone cells isolated from the Hyp mouse, the murine homologue for hypophosphatemic vitamin D-resistant rickets, produce abnormal bone when transplanted to either normal or phosphate-supplemented Hyp mice. To assess whether correction of the bone formation by mutant cells transplanted into either normal or Hyp mice could be achieved in the presence of supraphysiologic serum concentrations of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), recipient mice of both genotypes were infused continuously with 1,25-(OH)2D3 (0.2 μg/kg/day). Bone nodules present in transplants recovered after 14 days were characterized by measuring the osteoid thickness and volume. Administration of 1,25-(OH)2D3 to Hyp mice corrected the defective bone formation by normal cells but not by pair-transplanted Hyp cells, despite normalization of serum phosphate levels and 3-fold increases in serum 1,25-(OH)2D3. The osteoid thickness and volume in Hyp transplants into 1,25-(OH)2D3-treated Hyp mice were, however, markedly reduced down to values observed for Hyp transplants into recipient normal mice. Administration of 1,25-(OH)2D3 to normal mice improved further bone formation by mutant cells without affecting that by pair-transplanted normal cells. Administration of 24,25-(OH)2D3 (1 μg/kg/day) combined with 1,25-(OH)2D3 to recipient mice of both genotypes prevented the sharp fall in serum 24,25-(OH)2D3 but was not more beneficial than 1,25-(OH)2D3 alone for improving bone formation by transplanted Hyp cells. These observations demonstrate an abnormal response of the mutant cells to the extracellular environment and support the concept of an intrinsic osteoblast defect in the Hyp mouse.
Expression and functional role of nitric oxide synthase in osteoblast-like cells
Authors: Riancho José A. , Salas Eduardo , Zarrabeitia María T. , Olmos José M. , Amado José A. , Fernández-Luna José L. , González-Macías Jesús
Published: February 2010

Nitric oxide synthases (NOS) are enzymes that produce nitric oxide (NO) from L-arginine in a reaction yielding citrulline as a coproduct. Nitric oxide modulates the activity of a wide variety of cells, but little is known about its effects on bone cells. In the present study we report that the NOS inhibitor NG-monomethyl-L-arginine (NMMA) induced a dose-dependent inhibitory effect on the proliferation of the osteoblast-like cell lines MG63 and ROS 17/2.8. The inhibitory effect was prevented by increasing L-arginine concentrations in the medium and by the NO donor sodium nitroprusside. Likewise, NMMA inhibited interleukin-6 secretion, independently of its effect on cell number. NOS expression by MG63 cells was confirmed by measuring their ability to metabolize radiolabeled L-arginine to citrulline. NOS bioactivity was detected in unstimulated cells, but was markedly increased by stimulating the cells with cytokines, lipopolysaccharide, or 1,25-dihydroxyvitamin D3. NOS activity was partially dependent upon the presence of calcium in the medium. Furthermore, constitutive-type NOS (c-NOS) and inducible-type NOS (i-NOS) mRNA expression was detected in ROS 17/2.8 cells after reverse transcription and polymerase chain reaction amplification. In conclusion, osteoblast-like cells express c-NOS and i-NOS, and NOS activity seems to play an important role in the regulation of cell proliferation and function.
Discriminative value of biochemical markers of bone turnover in assessing the activity of Paget's disease
Authors: Alvarez Luisa , Guañabens Nuria , Peris Pilar , Monegal Ana , Bedim Jose L. , Deulofeu Ramon , de Osaba M. Jesus Martinez , Muñoz-Gomez Jose , Rivera-Fillat Francisca , Ballesta Antonio M.
Published: February 2010

Clinical biochemical markers of bone turnover are usually increased in Paget's disease. However, the analysis of “new” markers, such as serum bone alkaline phosphatase (BAP), carboxy-terminal propeptide of type I procollagen (PICP), tartrate-resistant acid phosphatase (TRAP), telopeptide carboxy-terminal propeptide of type I collagen (ICTP), and urinary pyridinoline (PYR) and deoxipyridinoline (D-PYR), may improve the diagnostic efficacy and the evaluation of Paget's disease compared with conventional markers, such as serum total alkaline phosphatase (TAP) and urinary hydroxyproline (HYP). To evaluate the diagnostic accuracy and the changes of biochemical markers of bone turnover according to Paget's disease activity, we measured the levels of all these markers in three groups of pagetic patients classified according to their serum TAP activity: G-I, patients with serum TAP lower than 250 U/l (upper limit) (n = 15); G-II, patients with serum TAP between 251 and 500 U/l (n = 18); and G-HI, patients with serum TAP greater than 501 U/l (n = 26). Serum TAP and BAP showed the highest diagnostic accuracy among the markers of bone formation with a sensitivity of 78% and 84%, respectively, when the specificity was 100%. Urinary PYR was the most sensitive marker of bone resorption. Also, urinary PYR showed the highest proportion of increased values in pagetic patients (73%) compared with urinary HYP (64%), urinary D-PYR (60%), serum ICTP (41%), or serum TRAP (39%). In pagetic patients with normal serum TAP activity (G-I), serum BAP concentration was increased in 60% of patients, and urinary PYR was increased in 40% of patients. However, when serum TAP was greater than 500 U/l (G-III), almost all markers of bone formation and resorption were increased. These results suggest that when Paget's disease activity is high, most biochemical markers of bone turnover are increased. However, when the disease activity is low, serum BAP and urinary PYR improve the detection of such disease. Moreover, the determination of serum TAP activity and, to a similar extent, serum BAP concentration associated with the measurement of urinary PYR excretion provides the best biochemical approach for assessing Paget's disease activity.
Relations between histologic indices of bone formation: Implications for the pathogenesis of spinal osteoporosis
Authors: Parfitt A.M. , Villanueva A.R. , Foldes J. , Rao D. Sudhaker
Published: February 2010

Wall thickness, a major determinant of trabecular thickness, falls with age and falls further in osteoporosis. To estimate the importance of defective osteoblast recruitment in the pathogenesis of this defect, we compared various histologic indices of bone formation in iliac bone biopsies in three groups of subjects—healthy premenopausal women, healthy postmenopausal women, and patients with postmenopausal osteoporosis and at least one nontraumatic vertebral compression fracture. Indices that reflect the frequency of activation of bone remodeling and consequent birth rate of new teams of osteoblasts (osteoid surface, mineralizing surface, osteoblast surface, and bone formation rate, all expressed per unit of bone surface) were each higher in healthy subjects who were postmenopausal than in those who were premenopausal, but lower in osteoporotic than in normal postmenopausal women. In each group, the primary surface measurements were significantly correlated with each other, but the correlation was less close in those with osteoporosis. Indices that reflect the average collective performance of individual teams of osteoblasts (mineralizing surface and osteoblast surface per unit of osteoid surface, mineral apposition rate, adjusted apposition rate, and wall thickness) were all lower in postmenopausal than in premenopausal normal subjects, and even lower in those with postmenopausal osteoporosis. The parameters of the regression lines relating bone formation rate to osteoblast surface were essentially the same in each group, indicating that bone formation rate per unit of osteoblast surface was unaffected by age or menopause, and was the same in osteoporosis as in healthy subjects of similar age. We conclude that postmenopausal osteoporosis is characterized by a partial reversal of the menopause-related increases in histologic indices of bone formation that reflect activation frequency, and by an exaggeration of the age-related declines in the indices that reflect osteoblast team performance. Defective osteoblast recruitment makes a major contribution to the fall in wall thickness; this single defect can account for much of the age- and disease-related decline in osteoblast team performance.
Bone marrow cells are targets for the anabolic actions of prostaglandin E2 on bone: Induction of a transition from nonadherent to adherent osteoblast precursors
Authors: Dr. Scutt Andrew , Bertram Petrina
Published: February 2010

Although prostaglandin E2 (PGE2) is known to stimulate bone formation in vivo, its mechanism of action is not well understood. Circumstantial evidence suggests that bone marrow cells (BMC) may well be involved in this, and in order to investigate this further we have studied the effect of PGE2 on proliferation and matrix synthesis in high-density BMC cultures and on colony-forming unit (CFU-f) formation efficiency by BMC in vitro. High-density cultures of BMC formed a collagenous, calcified matrix, synthesized osteocalcin and expressed alkaline phosphatase activity. The addition of PGE2 caused a concentration-dependent increase in total (but not specific) APase activity, cell number, and collagen accumulation. It was found that PGE2 need only be present during the first 48 hours of the culture period and that longer exposure had no additional effect PGE2 also caused a concentration-dependent increase in CFU-f formation, and it was found that this was due to the recruitment of new mesenchymal precursor cells from the nonadherent fraction of the BMC. Once again, the presence of PGE2 for only the first 48 hours of the culture period was enough to precipitate a maximal response. We conclude that one mechanism for the anabolic actions of PGE2 may be the recruitment of OB precursors from a population of nonadherent mesenchymal precursor cells present in the bone marrow.
Shared phenotypic expression of osteoblasts and chondrocytes in fracture callus
Authors: Hughes Steven S. , Hicks David G. , O'Keefe Regis J. , Hurwitz Shepard R. , Crabb Ian D. , Krasinskas Alyssa M. , Loveys Luke , Puzas Edward J. , Rosier Randy N.
Published: February 2010

Endochondral ossification in fracture healing of rats at 4, 8, 11, 14, and 21 days was analyzed using immunological and molecular probes for markers of the chondrocyte and osteoblast phenotype. These markers were osteocalcin, type I and type II collagen, including the probes homologous to the alternatively spliced forms of α1 type II collagen, type IIA and type IIB. Histologic examination was performed on serial sections of the same tissue blocks to correlate cellular morphology with the immunohistochemical and in situ hybridization findings. At the junction of the cartilaginous and osseous tissue, an overlap of phenotype and morphology was noted. At the 8-day time point, the cells with chondrocyte morphology expressed intracellular message for osteocalcin and type I collagen. Immunohistochemical analysis of these cells also demonstrated intracellular osteocalcin. However, high levels of the type IIA collagen mRNA, which has previously been associated with less differentiated mesenchymal precursor cells, were expressed in both chondrocytes and osteoblasts. At the later time point (21 days) there was a substantial decrease in the number of cells displaying shared phenotypic characteristics. In situ hybridization and immuno-histochemistry have permitted identification of an overlapping or shared phenotype in osteoblasts and chondroblasts in fracture callus. The findings raise important questions regarding the possible plasticity of mesenchymal cell phenotypes within the dynamic environment of fracture healing. Additional examination of these issues will further define factors involved in origin, differentiation, and maturation of bone and cartilage cells.
Decreased mineralization and increased calcium release in isolated fetal mouse long bones under near weightlessness
Authors: Dr. Van Loon Jack J.W.A. , Bervoets Dirk-Jan , Burger Elisabeth H. , Dieudonn É Suzanne C. , Hagen Jan-Willem , Semeins Cor M. , Doulabi Behrouz Zandieh , Veldhuijzen Paul J.
Published: February 2010

Mechanical loading plays an important role in the development and maintenance of skeletal tissues. Subnormal mechanical stress as a result of bed rest, immobilization, but also in spaceflight, results in a decreased bone mass and disuse osteoporosis, whereas supranormal loads upon extremities result in an increased bone mass. In this first in vitro experiment with complete fetal mouse cartilaginous long bones, cultured under microgravity conditions, we studied growth, glucose utilization, collagen synthesis, and mineral metabolism, during a 4-day culture period in space. There was no change in percent length increase and collagen synthesis under microgravity compared with in-flight 1× gravity. Glucose utilization and mineralization were decreased under microgravity. In addition, mineral resorption, as measured by 45Ca release, was increased. These data suggest that weightlessness has modulating effects on skeletal tissue cells. Loss of bone during spaceflight could be the result of both impaired mineralization as well as increased resorption.
Characterization of the mouse tartrate-resistant acid phosphatase (trap) gene promoter
Authors: Reddy S.V. , Hundley J.E. , Windle J.J. , Alcantara O. , Linn R. , Leach R.J. , Boldt D.H. , Roodman G.D.
Published: February 2010

Tartrate-resistant acid phosphatase (TRAP) is an iron-binding protein that is highly expressed in osteoclasts. To characterize the regulation of TRAP gene expression, progressive 5′ and 3′ deletions of a 1.8 kb fragment containing the 5′-flanking sequence were fused to a luciferase reporter gene. Two nonoverlapping regions of this 1.8 kb fragment had promoter activity. The upstream promoter (P1) was located within the region from –881 bp to –463 bp relative to the ATG, while the downstream promoter (P2) was located between –363 bp to –1 bp in a region we have previously shown to be an intron in transcripts originating from the upstream promoter. A putative repressor region for the P2promoter at –1846 bp to –1240 bp and a putative enhancer region at –962 bp to –881 bp relative to the ATG were identified. PCR analysis of promoter-specific transcription of the TRAP gene in various murine tissues showed that both promoters were active in several tissues. Transferrin-bound iron increased P1promoter activity 2.5-fold and hemin decreased P1promoter activity, but neither had any effect on P2 activity. These data show that the transcriptional regulation of the TRAP gene is complex and that iron may play a key role in TRAP gene regulation.
Expression and Regulation of Na-Dependent Pi Transport in Matrix Vesicles Produced by Osteoblast-like Cells
Authors: Montessuit C. , Bonjour J.P. , Caverzasio J.
Published: February 2010

Extracellular matrix vesicles (MV) are the loci of initial mineralization in several calcifying tissues. We recently reported that MV isolated from chicken epiphyseal cartilage are equipped with a Na-dependent Pi transport (NaPiT) system. The activity of the NaPiT system appeared to be crucial for the development of MV-mediated calcification. In the present study we investigated the expression of NaPiT activity in MV produced by the osteoblast-like cells MC3T3-E1. The relationship between changes in NaPiT activity in the intact cells and in the released MV was also examined. NaPiT activity in MV harvested from cultured MC3T3-E1 cells was transiently expressed. It was markedly increased between Days 8 and 10 (5- to 6-fold), and then gradually decreased. NaPiT activity was enriched in MV as compared with the parent osteoblast-like cells, while the Na-dependent transport system for alanine (NaAIaT) was not. When NaPiT activity was enhanced in osteoblast-like cells by fetal calf serum (FCS) or Pi depletion, Pi transport stimulation was observed in the derived MV as well. Alkaline phosphatase (AP) was differentially expressed and regulated in MV from MC3T3-E1 cell cultures, as compared with NaPiT. In contrast to the transient expression of NaPiT, AP activity in MV increased continuously with time in culture. It was stimulated by FCS treatment of the parent cells, but decreased in MV obtained from Pi-depleted cultures. These results suggest that the presence in osteogenic cells of selective regulatory mechanisms for the insertion and enrichment of Pi transport activity in released MV. This process is probably of biological significance for the regulation of the cascade of events that leads to the mineralization of the bone matrix.
Effects of endothelin-1 on Ca2+ signaling and secretion in parathyroid cells
Authors: Fujii Yoshio , Tomić Melanija , Stojilković Stanko S. , Iida Toshihiko , Brandi Maria Luisa , Ogino Yoshio , Dr. Sakaguchi Kazushige
Published: February 2010

It has been previously reported that parathyroid cells express endothelin (ET) receptors and secrete ET-1 in an extracellular Ca2+ concentration ([Ca2+]e)-dependent manner. Here, we examined the effects of ET-1 on intracellular signaling and parathyroid hormone (PTH) secretion in dispersed bovine parathyroid (bPT) cells, which comprise several cell types including epithelial and endothelial cells, in two cell lines, the rat parathyroid epithelial (PT-r) and the bovine parathyroid endothelial (BPE-1) cells. An RNA-polymerase chain reaction analysis revealed that both ETA and ETB receptors are expressed in bovine parathyroid tissue and BPE-1 cells, and only the ETA receptor is expressed in PT-r cells. PT-r cells also expressed an inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) receptor, and ionomycin induced an increase in the intracellular Ca2+ concentrations ([Ca2+]i) in a Ca2+-deficient medium, indicating the presence of an operative intracellular Ca2+ pool in these cells. In cells bathed in 1 mM [Ca2+]e, ET-1 induced a rapid and transient increase in the Ins(1,4,5)P3 production, which was associated with a similar profile of increase in [Ca2+]i and with a peak response of about 800 nM. No changes in the profile of [Ca2+]i responses were observed in ET-1–stimulated cells in the presence of Ca2+ channel blockers, or in Ca2+-deficient medium, indicating that Ca2+ mobilization was not associated with Ca2+ entry. Furthermore, a sustained stimulation with ET-1 induced a decrease in [Ca2+]i below the prestimulatory level in a large population of cells, and the percentage of the cell population that shows the sustained decrease of [Ca2+]i increased in higher ET-1 concentrations. [Ca2+]i in PT-r cells was also controlled by a [Ca2+]e-dependent mechanism that changed [Ca2+]i from 28 to 506 nM in a 0.1–3 mM concentration range with an EC50 of 1.2 mM, which is comparable to that reported for bPT cells. In the same range of [Ca2+]e, PTH secretion from bPT cells was inhibited with an IC50 of 1 mM, and ET-1 increased PTH release in a dose-dependent manner but without affecting the IC50 for the [Ca2+]e-dependent inhibition. Thus, the parathyroid epithelial cells appear to respond to ET-1 in a unique way, and the ET autocrine system can be regarded as a possible mechanism to modulate the sensitivity of [Ca2+]e-dependent PTH release.
Surface-associated proteins from Staphylococcus aureus demonstrate potent bone resorbing activity
Authors: Dr. Nair Sean , Song Yu , Meghji Sajeda , Reddi Kris , Harris Malcolm , Ross Alistair , Poole Stephen , Wilson Michael , Henderson Brian
Published: February 2010

Staphylococcus aureus infections are associated with rapid bone destruction in conditions such as osteomyelitis, bacterial arthritis, and infected orthopedic implant failure. How this bacterium induces bone destruction has not been defined. In studies of the role of oral Gram-negative bacteria in periodontal pathology, we have established that cell surface-associated proteins (SAPs) are potent stimulators of bone resorption. The surface-associated components from S. aureus have now been isolated and demonstrated to be extremely potent stimulators of bone resorption in the murine calvarial bone resorption assay. Bone resorption appears to be due to proteins, is not the result of contamination with lipoteichoic acid or muramvl dipeptide, and is potently inhibited by indomethacin and can be completely blocked by high concentrations of interleukin-1 receptor antagonist or TN3–19.12, a neutralizing monoclonal antibody to murine TNF. The SAP fraction can stimulate fibroblasts or monocytes to release osteolytic cytokines, but only at high concentrations. Fractionation of the SAPs by high performance liquid chromatography demonstrated that a number of fractions were osteolytically active. The most active contained a heterodimeric protein of molecular weight 32–36 kD. The presence of this osteolytically active surface-associated fraction may account for the bone resorption associated with local infection with S. aureus.
Rapamycin: A bone sparing immunosuppressant?
Authors: Romero David F. , Buchinsky Farrel J. , Rucinski Boguslaw , Cvetkovic Mirjana , Bryer Haldon P. , Liang Xiao G. , Ma Yanfei F. , Jee Webster S. S. , Dr. Epstein Solomon
Published: February 2010

Immunosuppressant therapy is associated with osteoporosis both clinically, post-transplantation, and experimentally. In rats, cyclosporin A (CsA) and FK506 induce a state of high turnover rapid bone loss. After 14 days of administration in immunosuppressive doses, the more recently discovered immunosuppressant, rapamycin, resulted in no change of cancellous bone volume. A longer study over 28 days has now been carried out; contrasting the new drug with CsA and FK506. Sixty, 10-week-old Sprague–Dawley rats were randomly divided into five groups of 12 rats each. The first group served as an aging control. The remaining four groups received, by daily gavage, a combined vehicle placebo, CsA 15 mg/kg, FK506 5 mg/kg, and rapamycin 2.5 mg/kg, respectively. CsA- and FK506-treated rats, but not those treated with rapamycin, demonstrated high turnover osteoporosis with raised serum 1,25(OH)2D (p < 0.05) and elevated serum osteocalcin (p < 0.05). The trabecular bone area was decreased by 66% (p < 0.01) in the CsA group and 56% (p < 0.05) in the FK506-treated group compared with the control animals. The CsA- and the rapamycin-treated groups failed to gain weight and developed severe hyperglycemia (>20 mmol/l, p < 0.001) by day 14 but which largely resolved by day 28. Unlike the groups treated with CsA and FK506, rapamycin-treated rats had no loss of trabecular bone volume but there was increased modeling and remodeling and a decreased longitudinal growth rate. Rapamycin may thus confer a distinct advantage over the established immunosuppressants in not reducing bone volume in the short term. However, the increased remodeling may pose a problem with long-term use, and the decrease in longitudinal bone growth would make the drug unsuitable for growing individuals.
Normal human osteoblast-like cells consistently express genes for insulin-like growth factors I and II but transformed human osteoblast cell lines do not
Authors: Okazaki Ryo , Conover Cheryl A. , Harris Steven A. , Spelsberg Thomas C. , Riggs Lawrence B.
Published: February 2010

Insulin-like growth factors I (IGF-I) and II (IGF-II) are anabolic for osteoblastic cells. Although expression of IGF-I and IGF-II mRNA has been demonstrated in rodent osteoblastic cells, little is known about IGF gene expression in human osteoblastic cell models. In this study we characterized IGF-I and -II mRNA expression in (1) normal human osteoblast-like (hOB) cells, (2) a simian virus 40 immortalized hOB (HOBIT) cell line, and (3) human osteosarcoma cell lines SaOS-2, TE-85, MG-63, and U-2. Since cross-hybridization of IGF cDNA probes with ribosomal RNA obscures detection of some of the multiple IGF transcripts in human cells, we replaced Northern analysis with the more specific ribonuclease protection assay (RPA). We also used the reverse transcriptase-polymerase chain reaction (RT-PCR) to assess whether mRNAs were present at trace levels. IGF-I mRNA expression was consistently observed in normal hOB cells only and by both RT-PCR and RPA. Among IGF-I transcript variants, Ea IGF-I mRNA was more abundant than the Eb mRNA in normal hOB cells. Trace levels of IGF-I mRNA were variably detected in SaOS-2 and U-2 osteosarcoma cells when RT-PCR was performed, but we found no IGF-I mRNA in HOBIT, TE-85, or MG-63 cells. IGF-II mRNA was expressed in normal hOB, HOBIT, TE-85, and U-2 cells as assessed by either method. Trace levels of IGF-II mRNA were observed only in one of three SaOS-2 cell preparations and only by RT-PCR. IGF-II mRNA was absent in MG-63 cells. Thus, our data indicate that (1) normal hOB cells express both IGF-I and IGF-II mRNA and (2) transformation of human osteoblastic cells may alter IGF gene expression. These results provide a basis for selecting appropriate cell models for the study of IGFs in human bone.
IGF-II receptor number is increased in TE-85 osteosarcoma cells by combined magnetic fields
Authors: Fitzsimmons Robert J. , Ryaby James T. , Magee Frank P. , Baylink David J.
Published: February 2010

Human osteosarcoma–derived osteoblast-like cells, TE-85, were used to assess the effect of a low frequency alternating magnetic field in combination with a controlled static magnetic field (combined magnetic fields, CMF) on insulin-like growth factor receptor regulation. In our culture system, application of a 15.3 Hz CMF induces a calculated maximum electrical potential in the culture media of 10−5 V/m. Initial characterization of TE-85 cells demonstrated that (a) TE-85 cells contain both type I insulin-like growth factor (IGF-I) and IGF-II receptors and (b) dose dependence for IGF-stimulated cell proliferation were comparable to the affinities of the IGFs binding to membrane binding sites (i.e., receptors had dissociation constants in the low nanomolar concentration range). The studies with CMF exposure revealed that CMF treatment for 30 minutes increased the number of IGF-II receptors in a frequency-dependent manner without affecting the number of IGF-I receptors. The CMF-dependent increase in IGF-II receptor number was associated with a significant increase in the IGF-II dissociation constant. These results indicate that membrane receptor levels can be altered by short-term exposure to low-energy, low-frequency electromagnetic fields and suggest a potential biochemical mechanism for electromagnetic effects on bone formation and remodeling.
Differences in calcium absorption and kinetics between black and white girls aged 5–16 years
Authors: Abrams Steven A. , O'Brien Kimberly O. , Liang Lily K. , Stuff Janice E.
Published: February 2010

To assess racial differences in calcium (Ca) metabolism, we measured Ca absorption and kinetics in 89 girls (38 black and 51 white) aged 4.9–16.7 years using a dual-tracer stable isotope technique. We found significantly greater rates of fractional (0.44 ± 0.13 vs. 0.25 ± 0.08, p < 0.0001) and total calcium absorption (406 ± 142 vs. 234 ± 82 mg/day, p < 0.0003) in black than in white postmenarcheal girls. Fractional absorption of Ca was also greater in black than in white premenarcheal girls (039 ± 0.14 vs. 0.30 ± 0.10, p = 0.01). Fractional absorption of Ca was more closely correlated to Ca intake in white (r = –0.42, p = 0.002) than in black girls (r = –0.25, p = 0.14). Urinary Ca excretion was significantly lower in pre- but not postmenarcheal black girls than in white girls. Calcium kinetic values associated with bone calcium deposition were greater in black girls, indicating a greater rate of bone Ca deposition in both pre- and postmenarcheal black girls. These results suggest that the greater bone mass accumulated during childhood and adolescence in black than in white females is due, in part, to greater rates of Ca absorption in black girls.
Effects of the bisphosphonate YM175 on bone mineral density, strength, structure, and turnover in ovariectomized beagles on concomitant dietary calcium restriction
Authors: Motoie Hiroyuki , Dr. Nakamura Toshitaka , O'Uchi Naoto , Nishikawa Haruko , Kanoh Hiroyuki , Abe Tetsushi , Kawashima Hiroyuki
Published: February 2010

We evaluated the effect of YM175 on bone in ovariectomized beagles fed a calcium-restricted diet for 18 months. Groups 1 (n = 6) and 2 (n = 6) underwent sham operation, and groups 3–6 were ovariectomized at the age of 21 months. Group 1 was fed standard dog chow (calcium: 1.4%), and groups 2–6 were given a low calcium diet (0.14%). Groups 3 (n = 7), 4 (n = 7), 5 (n = 7), and 6 (n = 7) were given YM175 orally at doses of 0, 0.01, 0.1, and 1.0 mg/kg/day, respectively. At the end of the experimental period, bone mineral density values measured in the lumbar (L2) vertebra, the femoral neck, and the midfemur, were reduced in groups 2 and 3. Bone strength, measured by compression tests on L2 body and L3 cancellous core specimens and by bending tests on the femoral neck, also decreased. YM175 treatment dose-dependently increased the values of these parameters. However, the reduction in torsional stiffness in the midfemur was not completely prevented. In the L4 body, trabecular thickness decreased in group 2 and trabecular separation increased in group 3. YM175 treatment prevented these changes, and the values in group 6 were maintained at the same levels as those in group 1. Bone formation rates were increased in groups 2 and 3. YM175 treatment decreased these indices, but the reduction was incomplete even with the highest dose (group 6). These results demonstrate that, in our model, YM175 maintained the mass, structure, and mechanical properties of cancellous bone. Increased bone turnover was not completely prevented by the doses employed, but the balance between net resorption and formation in one remodeling cycle would have been equilibrated by YM175.
Intermittent cyclical etidronate treatment maintains the mass, structure and the mechanical property of bone in ovariectomized rats
Authors: Katsumata Takashi , Dr. Nakamura Toshitaka , Ohnishi Hideo , Sakurama Teruo
Published: February 2010

We examined the mechanical properties of rat bones treated with etidronate intermittent cyclic treatment (etidronate-ICT). Fifty Fisher rats, 7 months of age, underwent ovariectomy (OVX; n = 40) or sham operation (n = 10). The OVX rats were assigned to 4 groups injected with etidronate at the respective dose of 0, 2, 4, or 8 mg/kg of body weight (bw) 5 days each week for 2 weeks, followed by a 10-week period of no treatment. This regimen was repeated for 48 weeks. At the end of the treatment period, the ultimate bending strength and structural stiffness of the femoral midshaft in the OVX-alone group were not reduced compared with the values of the sham group, while those in the etidronate-treated groups were significantly larger than the values of the OVX group. The compressive stiffness and strength of the vertebral bodies specimens prepared from L3 and L5 vertebrae were markedly decreased in the OVX group compared with those in the sham group, but the values in the etidronate-treated groups were maintained at the same levels as those in the sham group. Mechanical properties at the tissue level, such as elastic modulus and material strength of the femoral cortex, were increased in etidronate-treated groups compared with the OVX group. The flexural modulus was also increased. The compressive elastic modulus of vertebral body in treated groups was preserved at the same level as that in the sham group. These results clearly demonstrate that in ovariectomized rats long-term etidronate intermittent cyclic treatment preserves the mechanical properties both at the whole bone and the tissue level.
A comparison of the anabolic effects of parathyroid hormone at skeletal sites with moderate and severe osteopenia in aged ovariectomized rats
Authors: Qi H. , Li M. , Dr. Wronski T.J.
Published: February 2010

Previous studies have shown that parathyroid hormone (PTH) stimulates bone formation and completely restores lost cancellous bone at skeletal sites with moderate osteopenia in relatively young ovariectomized (OVX) rats. The current study was designed to determine whether PTH has similar bone anabolic effects in aged OVX rats and to compare the bone restorative response to PTH at skeletal sites with moderate and severe osteopenia. Female Sprague-Dawley rats were subjected to sham surgery or bilateral ovariectomy at 3 months of age and maintained untreated for the first year after surgery to allow for the development of moderate vertebral osteopenia and severe tibial osteopenia in OVX rats. Groups of baseline control and OVX rats were sacrificed at the end of this pretreatment period. The remaining OVX rats were then treated for 10 weeks with vehicle, antiresorptive agents alone (estrogen, the bisphosphonate risedronate, or calcitonin) or PTH alone. Other groups of OVX rats were treated concurrently with PTH and each of the antiresorptive agents. As expected, the proximal tibia of baseline OVX rats exhibited severe cancellous osteopenia, whereas the first lumbar vertebral body was moderately osteopenic. Treatment of OVX rats with antiresorptive agents alone failed to restore cancellous bone at both skeletal sites, whereas treatment with PTH alone markedly stimulated bone formation and completely restored lost cancellous bone in the lumbar vertebra. PTH also stimulated bone formation in the severely osteopenic proximal tibia of OVX rats but only marginally restored lost cancellous bone, possibly due to an inadequate number of bone spicules to serve as a foundation for new bone formation. Concurrent treatments with PTH and antiresorptive agents did not augment cancellous bone to a greater, or lesser, extent than treatment with PTH alone. The positive results from the moderately osteopenic lumbar vertebra indicate that cancellous bone of aged OVX rats retains its ability to respond anabolically to PTH. However, the negative results from the severely osteopenic proximal tibia of aged OVX rats may provide insight into the failure of the skeletons of some osteoporotic patients to respond adequately to anabolic agents such as fluoride or PTH.
Reproducibility of DXA absorptiometry: A model for bone loss estimates
Authors: Fuleihan Ghada El-Hajj , Testa Marcia A. , Angell Jennifer E. , Porrino Nancy , Leboff Meryl S.
Published: February 2010

The enhanced precision of dual-energy X-ray absorptiometry (DXA) allows the detection of very small changes in bone mineral density (BMD). True clinical changes in BMD in patients must be evaluated with the appropriate error of variance. We evaluated the responsiveness of our measures to true bone loss using a statistical variance components model that characterizes the variability associated with error introduced by the machine, operator, and subjects. Our techniques were applied to data from a prospective study of BMD measurements on spine phantoms and on pre- and postmenopausal women performed on the same day or up to 4 weeks apart with DXA (QDR 1000W, Hologic). Our model determined that most of the error in measurements was introduced by operators' and subjects' variability rather than machine performance. The false-positive rates for true bone change are significantly reduced when the appropriate CV% is used to estimate the significance of bone loss over time. Our study underscores the need to use the appropriate precision error to evaluate the clinical significance of changes in bone mass in individual subjects over time.
Effects of resistance training on regional and total bone mineral density in premenopausal women: A randomized prospective study
Authors: Lohman Timothy , Going Scott , Hall Matthew , Ritenbaugh Cheryl , Bare Lisa , Hill Anabel , Houtkooper Linda , Aickin Mikel , Boyden Thomas , Pamenter Richard
Published: February 2010

This study was designed to assess the effects of 18 months of resistance exercise on regional and total bone mineral density (BMD) and soft tissue lean mass (STL) in premenopausal women aged 28–39 randomly assigned to an exercise or control group. Twenty-two exercise and 34 control subjects completed the 18-month training study. All subjects were previously inactive and untrained women. Initial, 5-, 12- and 18-month assessments were made of total and regional BMD and total and regional STL using dual energy X-ray absorptiometry. All subjects consumed a 500 mg/day elemental calcium supplement throughout the study. Initial Ca intake without supplement averaged 1,023 mg/day in total sample. Serum levels of bone osteocalcin and dietary assessments using 12 randomly assigned days of diet records were also completed. Muscular strength was assessed from both 1 repetition maximum (RM) testing of 10 weightlifting exercises and by peak torque for hip abduction/adduction and knee extension/flexion. Training increased strength by 58.1% based on 1 RM testing and by 33.8% based on isokinetic testing at 18 months versus baseline. BMD increased significantly above baseline at the lumbar spine for the exercise group at 5 months (2.8%), 12 months (2.3%), and 18 months (1.9%) as compared with controls. Femur trochanter BMD increased significantly (p < 0.05) in the exercise group at 12 months (1.8%) and 18 months (2.0%) but not at 5 months (0.7%) as compared with controls. No changes in total BMD, arm BMD, or leg BMD were found. There was a 20% increase in BGP in the exercise group as compared with controls at 5 months and this difference was maintained throughout the study. For STL, significant increases for total, arm, and leg were found at 5, 12, and 18 months for the exercise group versus control ranging from 1–6% over baseline. These results support the use of strength training for increasing STL and muscular strength with smaller but significant regional increases in BMD in the premenopausal population.
Assessment of spine bone mineral density in ovariectomized rats using DXA
Authors: Yamauchi Hideki , Kushida Kazuhiro , Yamazaki Kaoru , Inoue Tetsuo
Published: February 2010

Measurements of lumbar spine (L1–L6) bone mineral density (BMD) and bone mineral content (BMC) of Wistar rats were obtained by dual energy X-ray absorptiometry (DXA) (QDR-1000W, Hologic Inc., Waltham, MA) to estimate reproducibility and investigate age-related changes. In addition we evaluated the accuracy of the technique in female rats. The coefficients of variation (CV) for spine BMD measurements were found to range from 0.73–1.04 in vivo and from 0.36–1.56 in vitro. The in vitro measurements were performed in a 3 cm deep water bath to stimulate an equivalent tissue thickness. Spine BMC, measured in vivo and in vitro correlated closely with the subsequently determined ash weights (r2 = 0.87 and 0.97, respectively). We examined age-related spine BMD by DXA. A relatively constant increase in spine BMD was observed from 6 weeks to 22 weeks; spine BMD remained stable between 22 and 58 weeks. No peak was observed in spine BMD. To evaluate the effect of estrogen deficiency on animals of different ages, we measured spine BMD weekly in female rats subjected to ovariectomy (OVX) or sham operation at 8 and 23 weeks of age. The spine BMDs in each OVX rat were significantly lower than that of the controls. In the 23-week-old rats, bone loss was quite rapid for the first 3 weeks of observation and stable afterward. The BMD of 8-week-old OVX rats increased with body size. We conclude that DXA allows the observation of age-related changes in the spine BMD of rats with great precision. The data we have gathered using this method should prove useful for the assessment of experimental models of osteoporosis in rats.