bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of high extracellular calcium concentrations on phosphoinositide turnover and inositol phosphate metabolism in dispersed bovine parathyroid cells
Authors: Kifor Olga , Kifor Imre , Brown Edward M.
Published: February 2010

We previously showed that high extracellular calcium (Ca2+) concentrations raise the levels of inositol phosphates in bovine parathyroid cells, presumably via the G protein-coupled, “receptor-like” mechanism through which Ca2+ is thought to regulate these cells. To date, however, there are limited data showing Ca2+-evoked hydrolysis of phosphoinositides with attendant increases in the levels of the biologically active 1,4,5 isomer of inositol trisphosphate (IP3) that would be predicted to arise from such a receptor-mediated process. In the present studies we used HPLC and TLC, respectively, to quantify the high Ca2+-induced changes in various inositol phosphates, including the isomers of IP3, and phosphoinositides in bovine parathyroid cells prelabeled with [3H]inositol. In the absence of lithium, high Ca2+ dose dependently elevated the levels of inositol-1,4,5-trisphosphate [I(1,4,5)P3], with a maximal, 4- to 5-fold increase within 5 s; the levels of inositol 1,3,4-trisphosphate [I(1,3,4)P3] first rose significantly at 5–10 s and remained 5- to 10-fold elevated for at least 30 minutes. These changes were accompanied by reciprocal 29–36% decreases in PIP2 (within 5–10 s, the earliest time points examined), PIP (within 60 s), and PI (within 60 s). These results document that, as in other cells responding to more classic “Ca2+-mobilizing” hormones, the high Ca2+-evoked increases in inositol phosphates in bovine parathyroid cells arise from the hydrolysis of phosphoinositides, leading to the rapid accumulation of the active isomer of IP3. The latter presumably underlies the concomitant spike in the cytosolic calcium concentration (Cai) in parathyroid cells.
In vivo assessment of forearm bone mass and ulnar bending stiffness in healthy men
Authors: Myburgh Kathryn H. , Zhou Lin-Juin , Steele Charles R. , Arnaud Sara , Marcus Robert
Published: February 2010

The cross-sectional bending stiffness EI of the ulna was measured in vivo by mechanical resistance tissue analysis (MRTA) in 90 men aged 19–89 years. MRTA measures the impedance response of low-frequency vibrations to determine EI, which is a reflection of elastic modulus E and moment of inertia I for the whole ulna. EI was compared to conventional estimates of bone mineral content (BMC), bone width (BW), and BMC/BW, which were all measured by single-photon absorptiometry. Results obtained from the nondominant ulna indicate that BW increases (r = 0.27, p = 0.01) and ulnar BMC/BW decreases (r = −0.31, p ≤ 0.005) with age. Neither BMC nor EI declined with age. The single best predictor of EI was BW (r2 = 0.47, p = 0.0001), and further small but significant contributions were made by BMC (r2 = 0.53, p = 0.0001) and grip strength (r2 = 0.55, p = 0.0001). These results suggest that the resistance of older men to forearm fracture is related to age-associated changes in the moment of inertia achieved by redistributing bone mineral farther from the bending axis. We conclude that the in vivo assessment of bone geometry offers important insights to the comprehensive evaluation of bone strength.
Adhesion molecules in skeletogenesis: I. transient expression of neural cell adhesion molecules (NCAM) in osteoblasts during endochondral and intramembranous ossification
Authors: Lee Yun-Shain , Chuong Cheng-Ming
Published: February 2010

We report that neural cell adhesion molecules (NCAM) are expressed transiently in developing chicken osteoblasts during osteogenesis using immunostaining on cryostat sections. NCAM is strongly expressed in most osteoblasts along bone trabeculae that coincide with the presence of collagen I and alkaline phosphatase activity. In endochondral ossification, NCAM is highly expressed in osteogenic buds as seen in the epiphysis and diaphysis of tibia and vertebrae. In intramembranous ossification, NCAM is seen in osteogenic condensation of calvaria and in the periosteum of tibial diaphysis. The expression is transient because NCAM is not expressed in mesenchymal cells before osteogenic condensation and NCAM expression is lost in osteocytes in later stages. The staining pattern suggests that NCAM is present on the cell membrane of osteoblasts. Using a specific monoclonal antibody, the osteoblast NCAM is shown to contain polysialic acid, which is enriched in embryonic brain. Northern blot analysis using chicken brain NCAM cDNA as probes showed two major sizes of mRNA at 6.4 and 4.2 kb in calvarial mRNA as opposed to bands at 7.2, 6.4, and 4.2 kb in the brain. An immunoblot showed major proteins at Mr 165 and 110 kd, unlike brain NCAM, which are 180, 140, and 120 kD. That NCAM is involved in bone morphogenesis is consistent with the general hypothesis that NCAM plays pivotal roles in mesenchymal condensation, as shown in the formation of muscle, kidney, skin, and cartilage. The results establish NCAM as a cell surface molecule expressed transiently during osteoblast lineage. The implication that NCAM may mediate osteoblast interaction and regulate skeletal morphogenesis is discussed.
Absence of calbindin-D28 expression in nonclassical 1,25-dihydroxyvitamin D targets: Analysis by polymerase chain reaction
Authors: Dr. Walters Marian R. , Hunziker Willi
Published: February 2010

CaBP-D28 mRNA expression in rat heart, testis, and lung was assessed by polymerase chain reaction (PCR). The animal model used was the hyperinduced vitamin D-treated rat (100 ng 1,25-dihydroxyvitamin D subcutaneously, daily for 7 days). For the PCR studies, two pairs of 20 mer oligonucleotide primers (designated 1–4 according to their position on the coding strand, but with primers 3 and 4 in reverse orientation) derived from the rat CaBP-D28 cDNA sequence were tested in various combinations. Optimal conditions were established using a 1:100 dilution of cDNA from normal rat kidney. Bands of the predicted sizes of 869 (1, 3), 994 (1, 4), 725 (2, 3), and 850 (2, 4) nucleotide base pairs resulted, but with varying intensities: 2,4 ∼ 1,3 > 1,4 > 2,3. Repeat PCR (recycling after 1:100 dilution and readdition of reagents and primers with at least one different primer) provided strong additional amplification, particularly with the 1,4/2,4 combination. Under these conditions, mixing experiments showed that CaBP-D28 transcripts were detectable at 10−7-to 10−9-fold lower levels of expression than in D+ kidney. When RNA was isolated and cDNA generated from test tissues from 4 individual vitamin D-stimulated (D+) and vitamin D-deficient (D∼) rats, repeat PCR (1,4/2,4 primer combination) provided no evidence of significant CaBP-D28 mRNA expression in the nonclassic target tissues, in contrast to strong bands in both the D− kidney (undiluted) and D+ kidney (1:100 dilution) preparations. Thus, these data demonstrate that if CaBP-D28 mRNA is expressed in rat heart, testis, or lung, it is present at levels at least 107- to 109-fold lower than in kidneys of D+ rats.
Journal of Bone and Mineral Research
Authors: Burger Elisabeth H. , Klein-Nulend Jenneke , Veldhuijzen J. Paul
Published: February 2010

The use of hydrostatic pressure to apply mechanical stress to bone organ cultures is reviewed. Ossifying long bones and calvarial rudiments are sensitive to this type of stress. Intermittent hydrostatic compression of near physiologic magnitude (ICF) has anabolic effects on mineral metabolism in such rudiments, and continuous hydrostatic stress of high magnitude (CCP) has catabolic effects. The effects of ICF may be ascribed to shear stress generated at tissue interphases of different chemical and mechanical properties. Local factors, such as prostaglandins and growth factors, seem to be involved in the tissue response to mechanical stress.
Journal of Bone and Mineral Research
Authors: Oettmeier Ralf , Arokoski Jari , Roth Andreas J. , Helminen Heikki J. , Tammi Markky , Abendroth Klaus
Published: February 2010

All tissues of the joint are affected in some way in osteoarthritis because the joint is an interactively functioning unit. Our goal was to investigate the combined responses of articular cartilage and subchondral bone to altered loading conditions to improve our understanding of the physiology of these two components and, ultimately, the pathophysiology of osteoarthritis. A group of 20 female beagle dogs were divided pairwise into runners (n = 10) and controls (n = 10). The running training on a treadmill started at the age of 15 weeks, and during the following 40 weeks the running distance was gradually increased to 40 km/day with a 15° uphill inclination. With this daily running distance the beagles ran another 15 weeks. The samples for histology were taken from 11 different locations of the knee joint. Subchondral bone and articular cartilage histomorphometry was carried out in three different regions of the specimens (central, middle, and peripheral regions) using an image-analyzing system and an eyepiece graticule. In all regions of the articular cartilage, both the uncalcified and calcified cartilage showed slightly increased thickness in the runner dogs. The change was more evident in the peripheral and the central areas. The thickness of the subchondral bone plate tended to be higher in runners, too. Bone histomorphometric parameters showed significant signs of increased remodeling. The most notable change was the enlargement of the bone formation surface. The most intense remodeling was usually observed either centrally or peripherally in the articular surface. The strongest increase in trabecular bone volume and thickness of the cartilage was recorded in the femoropatellar area. These topographically confined and separate responses of the articular cartilage and subchondral bone to long-distance running are presumably adaptive processes that provide better congruence and biomechanical stability to the articulating bone ends. Under pathologic conditions these processes may initiate the subchondral etiopathogenesis of osteoarthritis by impairing the shock-absorbing capacity of the joint.
Journal of Bone and Mineral Research
Authors: Ferretti Jose L. , Spiaggi Eduardo P. , Capozza Ricardo , Cointry Gustavo , Zanchetta Jose R.
Published: February 2010

In femora from rats or mice, the area and moment of inertia (but not the wall-lumen ratio) of a diaphyseal section correlated with biomass and were determinants of the strength and stiffness of the integrated bone. In otter metacarpals, however, the geometric variable typically associated with body weight and mechanical ability of the integrated bone was the wall-lumen ratio (not the sectional moment of inertia). These differences may be associated with the meaningfulness of body density for natural selection in diving species like otters. A negative relationship between wall-lumen ratio and material modulus of elasticity in bones from the three species pointed out the impossibility of increasing diaphyseal thickness and stiffness at the same time. The results are compatible with the hypothesis that ecologic habits are selectively more important than phylogenetic relationships between species for the determination of bone mechanical properties in the upper vertebrates.
Journal of Bone and Mineral Research
Authors: Vico L. , Alexandre C.
Published: February 2010

Our knowledge of the adaptation of human bone microgravity remains poor despite long-term Russian spaceflights and the recent use of accurate techniques for bone mass measurements. The extent of bone deficits in the adaptation of the whole skeleton is not clear. At the tissue level, bone resorption and formation activities have been studied only in bones from rats after spaceflights lasting a few days to 3 weeks. In these animals, architectural features consistent with osteoporosis have been found in the proximal tibia. In pregnant animals the osteoclast population is increased at other skeletal sites. In areas of weight-bearing bones that are not protected by muscular insertions, bone resorption is not markedly altered after 7 days of spaceflight and bone formation is reduced. In areas of weight-bearing bones with muscular insertions and in non-weight-bearing bones, similar changes in bone cell activity are delayed. The severity of the response seems to vary with the location of the bone in the skeleton and its initial level of bone turnover. After 12.5 days the acute bone changes are less and no additional changes are observed after 21 days in space. We conclude that generalized bone deficits do not appear to be a consequence of microgravity but occur in localized areas according to the level of modeling and remodeling and of the support function of each bone at 1 g.
Changes in axial bone density with age: A twin study
Authors: Kelly Paul J. , Nguyen Tuan , Hopper John , Pocock Nicholas , Sambrook Philip , Eisman John
Published: February 2010

Bone mineral density in adult life, which is an important determinant of fracture risk, is determined by peak adult bone density, achieved in early adulthood and subsequent rates of change during adult life. Cross-sectional twin and family studies indicate that the majority of population variation in bone density may be explained by genetic factors. Although there is evidence for a genetic effect on peak bone mass, it is unknown whether there is a genetic effect on rates of changes in bone density with age. Changes in lumbar spine and femoral neck bone density determined by dual-photon absorptiometry (Lunar DP3) were examined in a cohort of monozygotic (MZ, n = 21, 3 male and 18 female pairs, median age, range, 46; 24–75 years) and dizygotic twins (DZ, n = 19; 43, 25–65 years). The median follow-up was 3 years (range 1.1–5.5 years), with each subject having at least two and up to four bone density assessments. In these twins, genetic factors determine variation in rates of change (% change/year) in lumbar spine bone density, rMZ = 0.93 and rDZ = 0.51, p < 0.02 (one tailed), and Ward's triangle, rMZ = 0–60. rDZ = 0.11, p < 0.05 (one tailed). Model-fitting analysis was also consistent with a genetic effect on rates of change in bone density at the trochanteric site, although such an effect was not shown at the femoral neck. These data demonstrate, for the first time, the possible existence of genetic determinants of rates of change in bone mineral density in adults.
Fluoride-stimulated [3H] thymidine uptake in a human osteoblastic osteosarcoma cell line is dependent on transforming growth factor β
Authors: Reed Berenice Y. , Zerwekh Joseph E. , Antich Peter P. , Pak Charles Y.C.
Published: February 2010

Controversy exists regarding the effect of fluoride on human osteoblast proliferation. To learn more of the cellular action of fluoride, we chose the clonal osteoblast cell line HOS TE85 as a model system. In these phenotypically osteoblast-like cells, sodium fluoride stimulated [3H]thymidine incorporation in a dose-dependent manner over the concentration range 1 × 10−5-2 × 10−4 M. The fluoride-induced stimulation of [3H]thymidine uptake was dependent on cell density, being optimal at subconfluent cell numbers. Stimulation of [3H]thymidine uptake was inhibited by anti-transforming growth factor β but not by antibody to insulin-like growth factor I or β2-microglobulin. Transforming growth factor β was shown to be a biphasic stimulator of [3H]thymidine uptake in HOS TE85, with maximal stimulation occurring at 0.5 nM transforming growth factor β. In the presence of fluoride the cells were more sensitive to stimulation by this growth factor, with maximum effect occurring at 0.1 nM. Fluoride did not increase mRNA for transforming growth factor β following either 8 or 24 h of exposure. We conclude that fluoride activates osteoblast proliferation by modulating the cellular sensitivity to transforming growth factor β, a known stimulator of bone growth.
Identification of a competitive binding component in vitamin D-resistant new world primate cells with a low affinity but high capacity for 1,25-dihydroxyvitamin D3
Authors: Gacad Mercedes A. , Adams John S.
Published: February 2010

Monkeys in a number of different New World primate genera express a form of compensated target organ resistance to steroid hormones, including 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Characterization of these phenotypes has previously relied upon the study of the 1,25-(OH)2D3-receptor (VDR) interaction in cultured dermal fibroblasts from affected primates. In this report, we show that three of these prototypic phenotypes can be faithfully reproduced in previously established cultured cell lines: B95–8, EBV-transformed B lymphoblasts from the marmoset (Callithrix jacchus), a New World primate with recognized vitamin D resistance; OMK, renal tubular epithelial cells from the owl monkey (Aotus trivergatus), a New World primate with an Old World primate-like VDR phenotype; and MLA144, transformed B lymphoblasts from a gibbon (Hylobates), an Old World primate that expresses the wild-type VDR phenotype. The rank order of specific nuclear uptake and binding of [3H]1,25-(OH)2D3 to the VDR was OMK ≥ ML A144 » B95–8. Despite a 7- to 9-fold difference in cellular VDR content according to ligand binding analyses, there was no discernible difference in the internalization constant Kin for specific cellular uptake of [3H]1,25-(OH)2D3 (0.12–0.26 nM) or in the quantity of VDR detected by immunoblot analysis. We now speculate that the discrepancy in VDR quantitation by binding and immunoblot analysis in the B95–8 New World primate cell line results from the presence of an intracellular, vitamin D metabolite binding moiety in this cell line that competes with the VDR for metabolite binding. Extracts of B95–8 cells, but not wild-type MLA144 cells, exhibited a binding component that had a relatively low affinity (∼ 50 nM) but high capacity for 1,25-(OH)2D3 compared to the VDR. When mixed with extracts of OMK and MLA144 cells (containing the wild-type VDR) of equivalent protein concentration, B95–8 cell extract inhibited specific 1,25-(OH)2D3-VDR binding 84–86%. Sephadex G-100 gel filtration chromatography of the sterol binding component in the B95–8 cell extract detected a single peak of [3H]1,25-(OH)2D3 and [3H]25-OHD3 binding to a protein eluting with an apparent molecular mass of 58–60 kD. This 58–60 kD sterol binding component was also distinguished from the 50 kD VDR by its elution from DEAE-cellulose anion-exchange HPLC at 0.45 M KCl; the mammalian VDR eluted at 0.25 M KCl. These data suggest that the 1,25-(OH)2D3-resistant phenotype of B95–8 cells may be due to the presence of a relatively plentiful, 58–60 kD intracellular protein that binds 1,25-(OH)2D3 with low affinity but high capacity and inhibits the high-affinity nuclear localization of 1,25-(OH)2D3 to the VDR.
In vitro exposure to sodium fluoride does not modify activity or proliferation of human osteoblastic cells in primary cultures
Authors: Chavassieux Pascale , Chenu Chantal , Valentin-Opran Alexandre , Delmas Pierre D. , Boivin Georges , Chapuy Marie Claire , Meunier Pierre J.
Published: February 2010

The anabolic effects of sodium fluoride (NaF) on trabecular bone mass in osteoporosis is now well established. In vivo histologic studies performed in humans and other animals have shown that fluoride induces an increase in osteoblast number at the tissue level. To determine the mechanisms of action of fluoride on osteoblasts, we studied the effects of NaF on short- and long-term cultures of human osteoblastic cells derived from bone explants obtained from 21 donors. In short-term experiments, bone-derived cells were exposed to NaF for 4 days. At doses ranging from 10−11 to 10−5 M, NaF did not modify the alkaline phosphatase (AP) activity or osteocalcin secretion. In long-term experiments, half the bone samples from 15 donors were cultured for 4 months in the presence of 10−5 M NaF and the other half were maintained in NaF-free medium. Observations by light and electron microscopy disclosed no morphologic modification in bone ex-plants after 4 months of exposure to NaF, despite an increase in the bone fluoride content. After the first month of culture, slight but not significant increases were noted in 6 of 10 cases for AP activity, 4 of 10 for osteocalcin secretion, and 5 of 7 for [3H]thymidine incorporation. After 4 months of culture in the presence of NaF, no change in AP activity or cell proliferation was noted. In contrast, the osteocalcin secretion significantly decreased (p < 0.05). These data suggest that, in vitro, under the conditions of this study, there is no direct effect of fluoride on the proliferation or activity (AP activity and osteocalcin secretion) of human osteoblastic cells and that this effect is very likely mediated by a cofactor.
Immunolocalization of noncollagenous bone matrix proteins in lumbar vertebrae from intact and surgically menopausal cynomolgus monkeys
Authors: Carlson Cathy S. , Tulli Hermina M. , Jayo Manuel J. , Loeser Richard F. , Tracy Russell P. , Mann Kenneth G. , Adams Michael R.
Published: February 2010

The noncollagenous matrix proteins, composing about 10% of the organic matrix of bone, are considered important for cell matrix organization and regulation of mineralization in bone. In the present study, seven of the major noncollagenous bone matrix proteins were localized immunohistochemically in serial sections of lumbar vertebrae from 24 (12 intact and 12 ovariectomized) adult female cynomolgus monkeys (Macaca fascicularis). Osteocalcin was the only protein restricted to bone cells and mineralized bone matrix. Bone sialoprotein was present in both bone and calcified cartilage, and all the other proteins were distributed in soft tissues as well as bone. Staining for both osteocalcin and bone sialoprotein was present diffusely throughout the bone matrix, but osteonectin, osteopontin, matrix gla protein, decorin, and biglycan staining was concentrated along bone surfaces. Osteoid was negative for osteocalcin and bone sialoprotein, but all other proteins had areas of positive immunostaining within osteoid. All proteins except biglycan exhibited strong immunostaining of a subset of active osteoblasts, suggesting that they may be markers of osteoblast maturity or state of activation. The pattern of immunostaining in intact and surgically menopausal monkeys was similar, except that staining for matrix proteins concentrated along bone surfaces appeared to be more widely distributed in the surgically menopausal monkeys, probably due to the higher rate of bone formation in these animals.
Pseudohypoparathyroidism with osteitis fibrosa cystica: Direct demonstration of skeletal responsiveness to parathyroid hormone in cells cultured from bone
Authors: Murray Timothy M. , Rao Leticia Gomez , Wong Min-Min , Waddell James P. , McBroom Robert , Tam Cherk S. , Rosen Fred , Levine Michael A.
Published: February 2010

A young girl had tibial osteotomies at age 14 for genu valgum and then had recurrent tibia) cysts over a number of years. Hypocalcemia and hyperphosphatemia were first noted at age 21. The diagnosis of pseudohypoparathyroidism was made at age 28, when elevated plasma PTH was detected. Clinical and biochemical features, including a PTH response test and assay of RBC Gs, established the diagnosis of pseudohypoparathyroidism type lb. Failure to suppress plasma PTH with vitamin D therapy led to an exacerbation of her cystic bone disease; there were widespread lytic lesions radiologically, most of which took up [99mTc]diphosphonate on bone scan. Microradioscopy revealed evidence of resorption of phalangeal tufts. Bone biopsy showed osteitis fibrosa cystica. During an orthopedic procedure, trabecular bone fragments were taken from her right humerus, and bone-derived cells cultured using an explant technique. The cultured cells were osteoblast-like in morphology, fully responsive to PTH, cholera toxin, forskolin, and PGE1 in vitro, and had an alkaline phosphatase and osteocalcin response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Following this examination of skeletal responsiveness, attempts were made to suppress the elevated plasma PTH levels and symptomatic bone disease by optimizing therapy with oral 1,25-(OH)2D3. When bone pain associated with the cystic bone disease failed to resolve, the patient underwent total parathyroidectomy, following which the bone pain gradually resolved. This is the first direct demonstration of PTH responsiveness in cultured bone cells in the syndrome of pseudohypoparathyroidism with osteitis fibrosa cystica.
Detection of mRNA for carbonic anhydrase II in human osteoclast-like cells by in situ hybridization
Authors: Zheng Ming H. , Fan Ying , Wysocki Stan , Wood David J. , Papadimitriou John M.
Published: February 2010

Carbonic anhydrase II (CA II) plays an important role during osteoclastic bone resorption. Biochemical investigations of gene expression of CA II, however, have been hampered by difficulty in obtaining sufficient numbers of purified osteoclasts. In this study, we describe a nonradioactive, digoxigenin-labeled cDNA in situ hybridization technique capable of determining the pattern of CA II gene expression in human osteoclast-like cells (OC-like cells) at the single-cell level. The results showed that CA II mRNA was located in the cytoplasm of both imprinted and cultured OC-like cells from a giant cell tumor of bone. On the other hand, no evidence of CA II mRNA was found in either the mononuclear cells (tumor cells) of giant cell tumor of bone or osteosarcoma cells. There is a significant correlation between in situ hybridization and northern blot analysis for CA II mRNA in both the giant cell tumor of bone and the osteosarcoma. Our results also indicated that quantitation of in situ hybridization can be achieved by computed cytophotometry.
Proton channel part of vacuolar H+-ATPase and carbonic anhydrase II expression is stimulated in resorbing osteoclasts
Authors: Laitala Tuna , Väänänen Kalervo
Published: February 2010

Immobilization causes a transient increase in bone resorption and a prolonged depression of bone formation. We have studied the effect of immobilization on the expression of two proteins believed to have a major functional role in osteoclasts, the proteolipid subunit of vacuolar H+-ATPase (VPL) and carbonic anhydrase II (CA II). Trabecular bone from immobilized rat tibiae was analyzed using northern and slot blotting, polymerase chain reaction (PCR), and morphometric analysis. CA II and VPL transcription was rapidly stimulated in trabecular bone of immobilized rat tibiae. Osteoclast number increased and the trabecular bone volume decreased during immobilization. Fluorescein-labeled cDNA probes and a confocal laser scanning microscope were used to study the localization of VPL and CA II mRNAs in situ in osteoclasts and other bone-derived cells attached to bovine bone slices in vitro. CA II and VPL mRNA were highly expressed in actively resorbing osteoclasts, but in nonresorbing osteoclasts mRNA expression was very low or not detectable at all. These results strongly suggest that both CA II and VPL have an important functional role in bone resorption. Finally, in the bone cell population isolated for these studies, CA II was found to be highly specific for osteoclasts whereas VPL was also detected in other cell types.
A new method for vertebral fracture diagnosis
Authors: Ross Philip D. , Yhee Yhee K. , He Yi-Fan , Davis James W. , Kamimoto Carl , Epstein Robert S. , Wasnich Richard D.
Published: February 2010

A number of methods have been proposed for estimating the prevalence of vertebral fractures. Most methods are based on the distribution of normal vertebral dimensions in the population. However, these methods fail to identify a significant proportion (20–30% or more) of fractures documented on serial radiographs. This may occur because vertebral size varies between individuals as a result of differences in body size (and possibly other factors), and a normal range based on population reference data may be too large. In this paper, a new method is described for identifying vertebral fractures that are missed using diagnostic criteria based on vertebral dimension distributions of the population. This new method is based on calculating the average vertebral size, and statistical confidence limits, for the individual. The average vertebral size method was evaluated by testing its ability to identify incident fractures (which were identified from changes in dimensions compared to previous radiographs), using only the final film. The new method correctly identified most (81% of crush and 83% of wedge) incident fractures on the final radiograph. In contrast, criteria based on population distributions correctly identified only 53% of crush and 72% of wedge incident fractures. Using prospective data, prevalent fractures identified using both population-based and individual size-based criteria predicted the risk of incident fractures. Furthermore, incident fractures identified using both methods (population- and individual-based criteria) were associated with increased back pain. These data suggest that both types of prevalent deformities are important indicators of disease. We conclude that the new diagnostic method, which is based on an individual's vertebral size, will identify prevalent vertebral fractures missed by other methods and improve the accuracy of classification by fracture type (wedge, crush, and so on).
Improved treatment of paget's disease with dimethylaminohydroxypropylidene bisphosphonate
Authors: Schweitzer Dave H. , Zwinderman Aeilko H. , Vermeij Pieter , Bijvoet Olav L.M. , Papapoulos Socrates E.
Published: February 2010

A group of 89 patients with Paget's disease of bone were treated with different intravenous or oral doses of the nitrogen-containing bisphosphonate dimethylaminohydroxypropylidene bisphosphonate (dimethyl-APD). Biochemical remission was obtained in 82% of treatments, and in the rest a clear response was found. Oral dimethyl-APD was well tolerated, and a dose of 200 mg/day for 10 days was sufficient to induce remission in the majority of patients. The remission probability was 3 months for 50% of patients, and the recurrence-free period was 27 months. The remission probability as well as the recurrence-free period did not differ among oral and intravenous treatments or among patients who had never been treated with nitrogen-containing bisphosphonates and those who had received pamidronate in the past and were treated for a recurrence of the disease. Dimethyl-APD is a very effective bisphosphonate devoid of side effects, which given for a short period can induce long-lasting remissions in patients with Paget's disease of bone.
Differential expression of alkaline phosphatase in clones of human osteoblast-like cells
Authors: Dr. Manduca Paola , Sanguineti C. , Pistone M. , Boccignone E. , Sanguineti F. , Santolini F. , Federici A.
Published: February 2010

We established cultures of cells growing out from adult bone chips and maintained them through 12 passages in culture. The cultures showed osteoblastic phenotype accompanied by synthesis of collagen type I, osteonectin, alkaline phosphatase, and osteocalcin. We report the chracterization of 21 clones obtained from three different individual primary cultures. We studied the expression of osteonectin, alkaline phosphatase, collagen, and osteocalcin in the clones. Metabolic labeling showed production of type I collagen and of osteonectin in all clones studied. In two-thirds of the clones and in mass cultures alkaline phosphatase was not detected at passage 2, but it was detected in increasing amounts at later passages in culture. The clones attained different but detectable levels of expression of this marker by passage 8. The different levels in the expression of alkaline phosphatase in positive clones may be because they were derived from cells at different stages of osteoblastic maturation or due to small changes in microenvironment. The alkaline phosphatase-positive clones were tested for osteocalcin, and they showed measurable expression only at passage 10. A third of the clones obtained were negative for alkaline phosphatase during 12 passages in culture. The obtainment of clones unable to produce alkaline phosphatase may be due to loss of differentiating potential under the in vitro culture conditions. The growth rate and potential of all clones studied were similar through 12 passages in culture, regardless of their potential for expression of alkaline phosphatase.
Measurement of intrinsic bone quality in vivo by reflection ultrasound: Correction of impaired quality with slow-release sodium fluoride and calcium citrate
Authors: Antich Peter P. , Pak Charles Y.C. , Gonzales Jerome , Anderson Jon , Sakhaee Khashayar , Rubin Craig
Published: February 2010

The intrinsic (material) quality of cancellous and cortical bone was evaluated in vivo from the measurement of reflection ultrasound velocities in the ulna. In cancellous bone, the reflection ultrasound velocity was inversely correlated with age in normal women (r = −0.48, p = 0.001), with a significantly lower mean value in 32 normal postmenopausal women than in 14 premenopausal women (3124 versus 3341 m/s, p < 0.0001). In 32 untreated osteoporotic women the cancellous bone velocity was lower than in normal postmenopausal subjects (2906 versus 3124 m/s, p = 0.0001). Following treatment with slow-release sodium fluoride plus calcium citrate (mean 2.4 years in 33 osteoporotic patients with no fracture during treatment), the cancellous bone velocity was significantly higher than in untreated osteoporotic women (3082 versus 2906 m/s, p = 0.0002) and was not significantly different from that in normal postmenopausal women. The cortical bone velocity displayed similar trends, but the changes did not attain statistical significance. The measurements were repeated approximately 9 months later in 9 untreated and in 20 treated patients; in 5 additional patients, the measurements were made both before and after 9 months of treatment with slow-release sodium fluoride and calcium citrate. The cancellous bone velocity increased significantly (p = 0.046) in these patients, from 3008 m/s before treatment to 3112 m/s after the first 9 months of treatment. The velocity rose significantly from 3037 to 3167 m/s (p = 0.017) in patients treated for a short time (12–30 months at first measurement), but it did not change in untreated patients or those treated for more than 30 months. Thus, the material quality of cancellous bone decreases with normal aging and is reduced further with the osteoporotic process. This impaired quality may be corrected by treatment with slow-release sodium fluoride plus calcium citrate.
Effect of medium pH on osteoclast activity and osteoclast formation in cultures of dispersed rabbit osteoclasts
Authors: Shibutani Toshiaki , Dr. Heersche Johannes N.M.
Published: February 2010

We investigated the effect of medium pH on activity of isolated osteoclasts and have also looked at the possibility that medium pH affects osteoclast numbers during culture. Osteoclast-containing cell suspensions prepared from neonatal rabbits were cultured on bovine bone slices at pH 6.5, 7.0, or 7.5. After 24 or 48 h of culture, the cells and bone slices were fixed and stained for tartrate-resistant acid phosphatase (TRAP). After counting the osteoclasts, the cells were removed and the resorption lacunae stained by immunostaining using anticollagen type I antibody and then quantitated. We found that the resorptive activity of isolated rabbit osteoclasts was sharply increased at pH 6.5–7. Osteoclast differentiation and proliferation, on the other hand, were optimal at pH 7.0–7.5 but decreased at pH 6.5. The results thus imply that pH regulation of the bone surface environment can dramatically alter both the number of osteoclasts and their resorptive activity.
Bone disease in burn patients
Authors: Klein Gordon L. , Herndon David N. , Rutan Thomas C. , Sherrard Donald J. , Coburn Jack W. , Langman Craig B. , Thomas Mary L. , Haddad John G. , Cooper Cary W. , Miller Nancy L. , Alfrey Allen C.
Published: February 2010

Burn patients are at risk for bone disease due to aluminum (AI) exposure from use of antacids and albumin, partial immobilization, and increased production of endogenous glucocorticoids. Moreover, severely burned children are growth impaired up to 3 years after the burn. To determine the extent of bone disease, we studied nine men and three women, ages 18–41 years, with greater than 50% body surface area burn. Seven patients underwent iliac crest bone biopsy following double tetracycline labeling, one additional patient expired after a single label, and three others had postmortem specimens obtained for quantitative AI only. Serial serum and urine samples were obtained weekly until biopsy or death. All biopsied patients had reduced bone formation and osteoid area, surface, and width, with mineral apposition rate, osteoblast surface, and osteoclast number with normal eroded surfaces compared to age- and sex-matched normal ambulatory volunteers. Burn patients also had reduced bone formation, mineral apposition rate, osteoid area, and surface compared to age-matched volunteers at short-term bed rest. Serum levels of osteocalcin were low. Most patients had mild hypercalcemia but only a third had hypercalciuria. All patients had elevated AI in blood or urine; urine AI correlated inversely with serum osteocalcin. In 60% significant bone AI was detectable by stain or quantitation. Our data are compatible with burn patients having markedly reduced bone turnover. AI loading, partial immobilization, endogenous corticosteroids, and cytokine production may be among the etiologic factors.
Effect of ovariectomy on bone response to in vivo external loading
Authors: Hagino Hiroshi , Raab Diane M. , Kimmel Donald B. , Akhter Mohammed P. , Recker Robert R.
Published: February 2010

This study was undertaken to find whether the bone response to increased external loading (EL) of the tibia in rats is affected by estrogen depletion. Female Sprague-Dawley rats 6 months were randomly assigned to four groups of 10 each: sham ovariectomy without loading (Shm-XL), ovariectomy without loading (OVX-XL), sham ovariectomy with external loading (Shm-EL), and ovariectomy with external loading (OVX-EL). In vivo external loading by a four-point bending device was initiated 4 weeks after surgery. The right lower leg of each EL rat was loaded at 31.4 ± 0.2 N for 36 cycles at 2 Hz every other day for 21 days (11 loading days). Mean in vivo induced strain was 1305 microstrain (μϵ) for Shm-EL rats and 1280μϵ for OVX-EL rats. With external loading of the tibia, periosteal bone formation rose equally in Shm and OVX rats. Woven bone was present around the tibia or fibula in 60% of the loaded rats and none of the control rats. No loading response occurred either at the endocortical surface or in the cancellous bone of the proximal tibial metaphysis. After OVX, cancellous bone area in the proximal metaphysis declined and formation surface rose compared to Shm rats. Although periosteal and endocortical bone formation rose after OVX, no cortical bone loss occurred. We conclude that ovariectomy and attendant loss of endogenous estrogen do not change the cortical bone response to an external load of about 1300μϵ in rats. However, these results may not predict the cortical bone response to loading in animals with Haversian remodeling that display estrogen-related loss of cortical bone.
Molecular cloning and sequence analysis of cDNA for a 59 kD bone sialoprotein of the rat: Demonstration that it is a counterpart of human α2-HS glycoprotein and bovine fetuin
Authors: Ohnishi Tomokazu , Nakamura Osamu , Ozawa Masayuki , Arakaki Naokatu , Muramatsu Takashi , Daikuhara Yasushi
Published: February 2010

A complementary DNA (cDNA) for the 59 kD bone sialoprotein, which is supposed to be the rat counterpart of human α2-HS glycoprotein (α2-HSG) and is synthesized by both hepatocytes and osteoblasts, has been cloned from a rat liver cDNA library. Polyclonal rabbit antibodies to rat 59 kD bone sialoprotein were used to identify and isolate the cDNA. The amino acid sequence of 59 kD bone sialoprotein deduced from the cDNA revealed that the entire protein consisted of 352 amino acid residues, including a signal peptide of 18 amino acid residues, and contained three possible N-glycosylation sites. On Northern blot analysis of rat liver, an mRNA of about 1.5 kilobases was detected. An mRNA of 59 kD bone sialoprotein was also detectable in rat bone but not in other tissues, such as kidney, brain, and lung. A computer search of protein and nucleic acid data bases revealed that 68.2, 63.2, and 97.4% amino acid residues of 59 kD bone sialoprotein were identical with those of human α2-HSG, bovine fetuin, and rat phosphorylated N-glycoprotein (pp63), respectively. The positions of cysteine residues in 59 kD bone sialoprotein also completely matched those in human α2-HSG and bovine fetuin, indicating that the sialoprotein is the rat counterpart of human α2-HSG and bovine fetuin. In addition, comparison of the nucleotide sequence of cDNA for rat fetuin/α2-HSG with that for pp63 recently corrected showed only two differences in nucleotides in the entire protein coding regions of the two proteins, and immunoreactive rat fetuin/α2-HSG in the conditioned medium of adult rat hepatocytes in primary culture was found to be phosphorylated. Thus, because rat fetuin/α2-HSG isolated from bone and synthesized by osteoblasts in culture does not contain phosphorus, it seems to be pp63 dephosphorylated during circulation or in the bone matrix.
Insulin-like growth factor binding protein–4 inhibits both basal and IGF-mediated chick pelvic cartilage growth in vitro
Authors: Schiltz Patric M. , Dr. Mohan Subburaman , Baylink David J.
Published: February 2010

This laboratory has purified a unique insulin-like growth factor binding protein (IGFBP-4) that was previously demonstrated to be inhibitory to bone cell proliferation. In this study, the hypothesis that IGFBP-4 is inhibitory to insulin-like growth factor (IGF) actions on cartilage was tested using the pelvic cartilages of 10-day-old chick embryos as an in vitro model system. Pelvic leaflets were incubated in serum-free medium for 18 h with effectors (BSA, IGF-I, IGF-II, IGFBP-4, or a combination of IGF and IGFBP-4). After the first 8 h, 1.5 μCi [3H]thymidine per well was added. Cartilage growth was assayed by TCA-insoluble [3H]thymidine incorporation into DNA. Additional experiments were conducted under similar conditions to assess the actions of the effectors on cartilage dry weight over a 72 h time period. In separate experiments, serum-free medium conditioned by chick pelvic cartilages for 72 h was assayed for IGF-II by radioreceptorassay, IGF-I by radioimmunoassay, and IGFBP by western ligand analysis. Exogenous IGF addition increased [3H]thymidine incorporation and dry weight of cartilages compared to controls. IGFBP-4 decreased both parameters in basal cartilage growth and also inhibited IGF-mediated cartilage growth. Pelvic cartilages secreted in vitro both IGF-I and IGF-II and a 32–34 kD IGFBP. In conclusion, the IGFs are stimulatory to cartilage growth in vitro and embryonic chick cartilage in vitro produces both IGF-I and II as well as an IGFBP. Exogenous IGFBP-4 inhibits both the basal and IGF-mediated growth of chick cartilage, suggesting that IGFBP-4 acts to downregulate the growth-promoting effects of IGFs on cartilage growth.
Longitudinal study of bone mass in end-stage renal disease patients: Effects of parathyroidectomy for renal osteodystrophy
Authors: Copley John B. , Hui Siu L. , Leapman Stephen , Slemenda Charles W. , Johnston Conrad C.
Published: February 2010

The effectiveness of parathyroidectomy (PTHX) for the control of secondary hyperparathyroidism was assessed in 46 adult end-stage renal disease (ESRD) patients whose bone mineral content at the midshaft and distal radius was measured using single-photon absorptiometry (SPA) every 6 months before and after the surgery. They were compared to 46 age-, race-, and sex-matched ESRD patient controls who had not undergone surgery but who had had at least five SPA studies at similar intervals. Presurgery midradius bone mass was significantly lower for PTHX patients compared to controls. Comparing changes in bone mass of PTHX patients across surgery to controls in comparable time periods showed that PTHX patients lost significantly less bone mass after surgery. Similar results were obtained when rates of change in bone mass were evaluated. When patient characteristics were examined, the effect of surgery was found to be diminished in elderly patients and in oophorectomized patients. It is concluded that PTHX can have a salutary effect on renal osteodystrophy in the appendicular skeleton, but factors other than bone mass also need to be considered in identifying those patients who will benefit from surgery.
High-dose estrogen inhibits bone resorption and stimulates bone formation in the ovariectomized mouse
Authors: Bain Steven D. , Bailey Mason C. , Celino Darlene L. , Lantry Megan M. , Edwards Martin W.
Published: February 2010

In This Study, We Have Investigated Estrogen'S Capacity To Regulate Bone Formation And Resorption In The Ovariectomized Mouse, Evaluating The Dose And Site Dependence Of Estrogen Action On Bone Modeling And Remodeling Surfaces. To Quantitate Bone Resorption, The Skeletons Of Fifty 8-Week-Old Swiss-Webster Mice Were Prelabeled With [3H]tetracycline (3H-T) before initiation of treatment protocols. Ovariectomies (OVX) and sham surgeries were performed 3 days after the final 3H-T injection, and the animals were assigned to treatment groups and injected once per week for 4 weeks with one of the following doses of 17β-estradiol (E2): sham/oil vehicle (SV), OVX/oil vehicle, OVX/50 μg E2, OVX/250 μg E2, and OVX/500 μg E2. To assess bone formation, fluorochrome labels were administered 9 and 2 days before sacrifice. At the conclusion of the 4 week protocol, the femora and thoracic vertebrae were removed to quantitate the levels of bone resorption based on the skeletal retention of 3H-T. The tibiae were excised for histomorphometric evaluation of the proximal metaphyses and middiaphyses. Indicative of increased bone resorption, vehicle-treated OVX animals had significantly reduced levels of 3H-T in femora and vertebrae compared to SV mice. This result was consistent with histomorphometric data showing a 49% decrease in cancellous bone area of the proximal tibiae in the OVX/oil-treated group. Treatment of OVX animals with 50 μg E2 was sufficient to maintain 3H-T levels in vertebrae at SV values, with higher E2 doses leading to a dose-dependent increase in the retention of 3H-T at this site. In contrast, 3H-T levels in the femora of mice treated with 50 and 250 μg E2 were intermediate between the SV- and vehicle-treated OVX animals. Only at 500 μg E2 per week were 3H-T values in femora maintained at SV values compared to OVX mice treated with vehicle. Histology data indicated that treatment of OVX mice with 250 μg E2 prevented osteopenia in the proximal metaphyses of the tibiae; the 500 μg dose of E2 led to a fivefold increase in cancellous bone volume compared to SV mice. In OVX mice treated with 500 μg E2, cancellous bone formation rates were increased 83% compared to SV animals and 35% compared to the OVX/oil-treated group. These increases were a consequence of increased bone-forming surface. E2 treatment also stimulated bone formation on the endosteal surface of the diaphysis. In summary, these results indicate that E2 inhibits bone resorption in femora and vertebrae of OVX mice in a dose-dependent fashion and has the capacity to stimulate bone formation on both endosteal and cancellous bone surfaces.