bluuebunny/crossref_metadata_2025 huggingface dataset:
Journal of Bone and Mineral Research
Authors: Mitlak Bruce H. , Daly Margaret , Potts John T. , Schoenfeld David , Neer Robert M.
Published: February 2010

Of 118 consecutive white patients referred for asymptomatic primary hyperparathyroidism, the diagnosis was clinically confirmed in 100, of whom 85 adults had a serum calcium less than 3.0 mM (12 mg/dl) and no skeletal, rheumatic, or significant neuropsychiatric symptoms, azotemia, or other significant illnesses. Among these 85, 68 had both asymptomatic and medically uncomplicated hyperparathyroidism, whereas 17 had historical, radiographic, or ultrasonic evidence of renal stone disease. The 20% with past or present renal calculi concentrated their urine significantly better than the 68 others (p = 0.05), but these two groups were otherwise not distinguished by the tests we performed, so all 85 patients were analyzed together. Systolic and diastolic blood pressures were normal, but premature osteopenia and/or impaired renal function were present in 29–36% of the patients. Micrometer measurements of metacarpal radiographs and 125I photon absorptiometry at the shaft of the radius revealed cortical osteopenia. Osteopenia was equally significant in the distal radius (cortical plus trabecular bone). These quantitative measurements were superior to routine bone radiography, and ROC analysis showed that 125I absorptiometry at either site was superior (p < 0.01) to metacarpal cortex measurements for detecting premature osteopenia, which was present in more than a third of these patients. Creatinine clearances (24 h) and maximum urine concentrating capacity (overnight dehydration plus the synthetic vasopressin analog DDAVP) were each significantly reduced, despite all patients' normal BUN and serum creatinine levels. Sequential performance of a 24 h creatinine clearance and a urine concentration test revealed abnormalities in the renal function of 27 of 74 patients (36%), with a specificity of 95% and a higher sensitivity than either test alone (27–29%). Testing the kidneys' ability to acidify the urine to pH 5.3 or less identified only 1 additional abnormal patient. Silent complications of primary hyperparathyroidism are thus common in “asymptomatic” patients with mild hypercalcemia. Their presence was only weakly correlated with urinary calcium excretion (measured on a low-calcium diet) and serum alkaline phosphatase. From these data, it is not possible to predict whether these complications progress in the absence of treatment or diminish with successful treatment of the primary hyperparathyroidism.
Journal of Bone and Mineral Research
Authors: Clark Orlo H. , Wilkes William , Siperstein Allan E. , Duh Quan-Yang
Published: February 2010

Several recent articles question whether patients with asymptomatic hyperparathyroidism and minimal hypercalcemia should be treated by parathyroidectomy. We therefore reviewed our experience in 103 consecutive patients with primary hyperparathyroidism who were treated by parathyroidectomy to determine, first, how many of these patients had asymptomatic or symptomatic hyperparathyroidism, and second, did these patients benefit from parathyroidectomy? We also analyzed the safety of parathyroidectomy in 426 consecutive patients, including 79 who required reoperation for hyperparathyroidism, specifically looking for complications and the outcome of these procedures. Our study documents the following: (1) only 2 of 103 (2%) patients referred for parathyroidectomy had “true” asymptomatic hyperparathyroidism; (2) only symptoms of fatigue, bone pain, and weight loss correlated with the degree of hypercalcemia, whereas muscular weakness, psychiatric symptoms, nocturia, polyuria, recent memory loss, constipation, and nephrolithiasis did not; (3) only 1 of 15 patients who were referred as asymptomatic were truly asymptomatic after more thorough questioning, and all 14 improved following parathyroidectomy; (4) 81% of the patients who were referred with symptoms improved following parathyroidectomy; and (5) permanent complications occurred in only 4 patients. All but 1 had reoperations for persistent or recurrent hyperparathyroidism (3 vocal cord paralyses and 1 hypoparathyroidism requiring autotransplantation of cryopreserved parathyroid tissue). There was 1 death of an 84-year-old woman with hypercalcemic crisis. Thus, most patients with hyperparathyroidism are symptomatic and benefit symptomatically and metabolically from parathyroidectomy, which is a safe operation.
Mineral metabolism in infants with malignant osteopetrosis: Heterogeneity in plasma 1,25-dihydroxyvitamin D levels and bone histology
Authors: Cournot Giulia , Trubert-Thil Claire Lise , Petrovic Milka , Boyle Allison , Cormier Catherine , Girault Danielle , Fischer Alain , Garabedian Michèle
Published: February 2010

A group of 16 infants, 2 weeks to 11 months old, with malignant osteopetrosis were investigated to examine their vitamin D metabolism and parathyroid function. Bone biopsies from 6 children were studied by light microscopic histomorphometry and by electron microscopy. Considerable heterogeneity existed among the patients with respect to the parameters reflecting mineral metabolism and with respect to the histological manifestations of the disease. The most constant findings were as follows. Immunoreactive parathyroid hormone (iPTH) was elevated in all children, except in 1 patient who had tubular acidosis, and plasma calcium was low or normal, suggesting skeletal resistance to PTH. Plasma 1,25-dihydroxyvitamin D [1,25-(OH)2D] was not constantly elevated and appeared to depend on plasma phosphorus, as both parameters were negatively correlated (r = 0.704, p < 0.01). Osteoblast activity, as evaluated by circulating alkaline phosphatase and osteocalcin and osteoblast number, measured for 6 children by bone histology, were not increased, despite hyperparathyroidism, suggesting PTH resistance or defective osteoblasts. Osteoclasts could be detected in 5 of the 6 children who had a biopsy. Osteoclast number (5.7–13.3% of bone surface) was normal or mildly increased, and marrow spaces were relatively well developed in 4 patients, whereas 1 child had markedly increased osteoclast number (28.3% of bone surface) and reduced marrow cavities. These 5 children received transplants, and engraftment occurred in all, except in the “hyperosteoclastic” patient. Further studies are necessary to establish the prognostic significance of this histologic feature.
Glucocorticoids decrease vitamin D receptor number and gene expression in human osteosarcoma cells
Authors: Godschalk Michael , Levy James R. , Downs Robert W.
Published: February 2010

The mechanisms by which glucocorticoids (GC) inhibit some actions of vitamin D [1,25-(OH)2D3] are not well understood, but there is growing evidence that GC alter vitamin D receptor (VDR) number. We studied the effects of dexamethasone (DEX) on VDR number and mRNA in the human osteosarcoma cell line, MG-63. The effects of DEX on 1,25-(OH)2D3 binding were examined by incubating confluent cells overnight in media without or with 10−6 M DEX. DEX decreased VDR number (Bmax) by ˜ 70% (110 versus 32 fmol/mg cellular protein, p < 0.001) without significantly changing the apparent affinity (K'D) of 1,25-(OH)2D3 for its receptor (3.8 versus 2.2 x 10−10 M, p > 0.05). Overnight incubation of MG-63 cells with DEX produced a time- and dose-responsive decrease in VDR mRNA compared to untreated controls (p < 0.01). To determine the mechanism of the DEX-mediated decrease in VDR mRNA, the effect of DEX on VDR mRNA stability was studied. We found that the half-life for the VDR mRNA was ˜ 5.7 h and was not significantly changed when the cells were incubated with DEX ( ˜6.3 h). We conclude that DEX decreases both VDR number and mRNA in MG-63 osteosarcoma cells. Since the half-life of VDR mRNA was not significantly modified by dexamethasone, glucocorticoids appear to decrease VDR mRNA by inhibiting VDR gene transcription or by affecting the processing of VDR mRNA.
Dose-related effects of 1,25-dihydroxyvitamin D3 on growth, modeling, and morphology of fetal mouse metatarsals cultured in serum-free medium
Authors: Bagi Cedo M. , Miller Scott C.
Published: February 2010

A serum-free, fetal bone organ culture model that permits the simultaneous determination of modeling and growth parameters was used to examine the effects of a near physiologic and a pharmacologic dose of 1,25-dihydroxycholecalciferol [1,25-(OH)bD3]. The fetuses of pregnant mice were removed on day 17 of gestation, and three medial metatarsal rudiments were cleaned and after preculturing were cultured as pairmatched groups for 4 days in MEM supplemented with 0.2% BSA. 1,25-(OH)2D3 was added to the cultures at concentrations of 10−2 or 10−6 M. Cultures treated with the carrier and devitalized bones served as controls. For resorption studies, pregnant mice were given 45Ca on day 17 of pregnancy and fetal metatarsals harvested 24 h later. Resorption was determined by the amount of 45Ca released into the media. DNA synthesis was estimated by determining the incorporation of [3H]thymidine, collagen synthesis by measuring the incorporation of [3H]proline, mineralization by the incorporation of 45Ca, and proteoglycan synthesis by the incorporation of 35S. The amount of radiolabel was detected in media, as well as in noncultured, dead, and cultured rudiments. The total length of the rudiments and length of the calcified diaphyses were measured daily. In addition, rudiments from all experimental groups were prepared for light and electron microscopy. The high dose (10−6 M) of 1,25-(OH)2D3 suppressed total rudiment growth but not the growth of the calcified diaphysis. 1,25-(OH)2D3 also decreased DNA, collagen, and proteoglycan synthesis, reduced calcification, and increased bone resorption in a dose-related manner. There were morphologic and ultrastructural changes in the osseous tissues and cells, particularly with the high dose of vitamin D, that supported the biochemical findings of suppressed activity of the osteogenic and chondrogenic cells. However, the suppression of collagen production and bone cell proliferation observed with the pharmacologic dose of vitamin D may be partially attributable to the decrease in bone mass (from increased resorption), thus resulting in less osseous tissue surface for these events to occur as endochondral osteogenesis progressed. The lower dose of vitamin D, however, had effects on 35S and 45Ca incorporation that could not be attributed to a decreased osseous tissue mass. This study emphasizes the importance of measuring specialized activities of the various cell populations in bone rudiment culture models to more fully understand the changes in tissue metabolism that result in changes in rudiment growth and modeling.
Interaction of androgen and 1,25-dihydroxyvitamin D3: Effects on normal rat bone cells
Authors: Gray Colin , Colston Kay W. , Mackay Alan G. , Taylor M. Louise , Arnett Timothy R.
Published: February 2010

We studied the actions of testosterone (T) and 5α-dihydrotestosterone (DHT) in combination with 1,25-di-hydroxyvitamin D3 [1,25-(OH)2D3] on primary rat bone cells. The actions of androgens were generally anabolic, although response patterns varied considerably between cultures. For example, DHT caused striking dose- and time-dependent increases in [3H]thymidine incorporation into calvarial cells over the range 1–100 nM, with maximal stimulation of 2.5-fold after 9 days in culture. Testosterone (50 nM) also stimulated [3H]-thymidine incorporation into long bone-derived cells. 1,25-(OH)2D3, generally blunted or abolished the proliferative action of androgens but was not itself always inhibitory; in some experiments, stimulation of [3H]-thymidine incorporation occurred. Collagen production, as assessed by [3H]proline incorporation into pepsin-resistant protein secreted by calvarial cells, was also stimulated by DHT. In some cultures, androgen responses were absent, although striking inhibitory responses to 1,25-(OH)2D3 were observed. These results illustrate complex patterns of responses to androgens and 1,25-(OH)2D3 in cells derived from rat bone.
Bone mass and body composition in normal women
Authors: Lindsay Robert , Cosman Felicia , Herrington Brenda S. , Himmelstein Sharon
Published: February 2010

The interrelationships between measurements of bone mass and total-body bone mineral were examined in a cross-sectional study of normal healthy women aged 17–82 years. In addition we evaluated the relationship between measures of body composition, estimated by four independent techniques, and bone mass in the same population. Considering the group as a whole, bone mass at all sites correlated with each other and with total-body bone mineral (TBBM). Cancellous and cortical sites could predict TBBM equally well. As expected, all measurements of bone mass were significantly lower in postmenopausal women in comparison to premenopausal women. Declines in bone mass were only seen in premenopausal women in the femoral neck and Ward's triangle, not in lumbar spine, radius, or skeleton as a whole. In postmenopausal women bone mass correlated negatively with age and years from menopause equally at all sites. TBBM was significantly related to height and weight in both premenopausal and postmenopausal women. In premenopausal and postmenopausal women TBBM also correlated with fat mass, but TBBM was much better correlated with percentage body fat in premenopausal than postmenopausal women. TBBM was a constant proportion of lean body mass in premenopausal women, but the fraction of lean mass occupied by the skeleton declined with age in postmenopausal women. Correction of TBBM for lean mass did not change the relationship between TBBM and percentage fat in premenopausal women but eliminated the relationship in postmenopausal women. Regional measurements, which are at least partially corrected for body size by dividing mass by area, correlated less well with height and weight and with any index of obesity, especially in postmenopausal women.
Effects of continuous and intermittent administration and inhibition of resorption on the anabolic response of bone to parathyroid hormone
Authors: Hock J.M. , Gera I.
Published: February 2010

The role of resorption in the anabolic response of bone to parathyroid hormone (PTH) is not well understood. In contrast to the increase in bone mass induced by intermittent PTH in intact rats, continuous infusion of PTH into thyroparathyroidectomized (TPTX) rats failed to increase bone volume. The objectives of this study were to determine if continuous infusions of low doses of PTH were anabolic in intact rats and if inhibition of resorption would enhance or block an anabolic action of PTH. Young male rats were treated with either continuous infusion or intermittent injections of hPTH-(1-34) for 12 days. In experiment 1, PTH, infused daily at 4 μg per 100 g, increased femur calcium and dry weight. Unlike infusion of 8 μg PTH, which did not alter bone mass, intermittent PTH at 8 μg was anabolic and increased bone mass by increasing trabecular thickness and number. Infusion of 16 μg induced hypercalcemia and death. In experiment 2, lower dose daily infusions of 0.25–4 μg PTH per 100 g did not increase bone mass. In experiment 3, in rats pretreated with dichloromethylene diphosphonate (CI2MDP) to inhibit resorption and subsequently exhibiting decreased bone formation, PTH, irrespective of the method of administration, reversed the inhibitory effects of Cl2MDP on bone formation. Thus, intermittent and continuous PTH increase bone formation independently of effects on bone resorption, but only intermittent PTH increases bone mass consistently.
Effects of five daily 1 h infusions of alendronate in paget's disease of bone
Authors: O'Doherty D.P. , Gertz B.J. , Tindale W. , Sciberras D.G. , Survill T.T. , Kanis J.A.
Published: February 2010

We report a randomized placebo-controlled double-blind study of amino-hydroxybutylidene bisphosphonate (alendronate), infused over 1 h, in 15 patients with Paget's disease of bone. Alendronate, 10 mg/day for 5 days, suppressed urinary hydroxyproline to 44.9 ± 4.8% and serum alkaline phosphatase to 74.6 ± 5.4% of their pretreatment values within 1 month of the start of treatment. Within 5 months of the start of treatment serum alkaline phosphatase fell to 47.9 ± 6.3% of pretreatment values. These effects were associated with a decrease in serum calcium and phosphate and in urinary calcium excretion and with a rise in serum iPTH values. A transient fever was observed in 3 of 10 patients who received alendronate during the course of the infusions, and this was associated with a decrease in the total and differential white cell count. No adverse effects were noted on renal function as judged by glomerular filtration rate and indices of proximal and distal tubular function. This regimen may simplify the management of patients with Paget's disease of bone.
Lateral dual-energy radiography: Artifact error from rib and pelvic bone
Authors: Rupich Reta C. , Griffin Michael G. , Pacifici Roberto , Avioli Louis V. , Susman Noah
Published: February 2010

A lateral projection of the lumbar spine with dual-energy radiography (DER) may be a more sensitive tool for the detection of vertebral demineralization than the standard anterior-posterior (AP) DER measurement. The lateral view allows measurement of the vertebral body and selective measurement of the central trabecular compartment, excluding the majority of the cortical envelope and posterior vertebral elements. The lateral DER typically contains L2-4 in the region of interest. The present study was designed to investigate the frequency and bone mineral content (BMC) contribution of rib to the L2 vertebral body BMC measurement in a lateral view DER scan. In addition, the frequency of pelvic overlap of L4 was evaluated. All patients were scanned in the supine position. Abdominal CT images of L2 were examined in 20 women aged 28–82 years to determine the frequency of rib and pelvic interference. QCT images of L2 also were examined in an additional 35 women aged 20–82 years to further investigate the frequency of rib overlap and to calculate the potential contribution of rib superimposition to the L2 BMC in a lateral projection. Pelvic overlap of L4 occurred in 15% of the 20 cases reviewed. Rib overlapped the L2 vertebral body in 100% of the L2 images. The expected contribution of rib BMC to L2 BMC in a lateral DER scan was 10.4% on average. There was no relationship between the percentage of rib BMC contribution and age and relatively poor correlations between rib BMC and that of either the vertebral body or the central trabecular compartment of L2. We conclude that supine positioning allows routine inclusion of L3 and L4 in a lateral lumbar scan. Results also suggest that L2 should be excluded from DER scans on patients scanned in either the lateral decubitus or supine position.
Stimulation of bone formation in osteoporosis patients treated with fluoride associated with increased dna synthesis by osteoblastic cells in vitro
Authors: Marie Pierre J. , De Vernejoul Marie Christine , Lomri Abderrahim
Published: February 2010

In this study we evaluated whether the fluoride-induced increased bone formation in osteoporosis is mediated by stimulation of bone cell proliferation and/or differentiation. We analyzed the kinetics of DNA synthesis and the phenotypic features of osteoblastic cells isolated from the trabecular bone surface in relationship to histomorphometric indices of bone formation evaluated on the same bone biopsy in 12 osteoporotic patients treated with fluoride. Osteoblastic cells isolated from patients with a higher than normal bone formation rate, increased mean wall thickness of trabecular bone packets, and high trabecular bone volume after fluoride therapy displayed a higher than normal rate of DNA synthesis in vitro. The peak of [3H]thymidine incorporation into DNA, the maximal DNA synthesis, and the area under the growth curve of osteoblastic cells isolated from these patients were higher than the values in normal bone cells obtained from agematched controls. By contrast, in vitro parameters of osteoblastic cell proliferation were not different from normal in fluoride-treated osteoporosis patients in whom bone formation was not increased, although the duration of treatment and bone fluoride content were not different. Parameters of bone cell proliferation in vitro were increased in correlation with the mean wall thickness, and the latter correlated with the trabecular bone volume, indicating that the augmentation of bone formation and bone volume induced by fluoride was paralleled by an increased proliferation of osteoblastic cells. Basal osteocalcin production (corrected for cell protein) and alkaline phosphatase activity in vitro were comparable, and the response to 1,25-dihydroxyvitamin D3 (10 nmol/liter, 48 h) was not different in normal osteoblastic cells and in cells from fluoride-treated osteoporosis patients whether they had high or normal bone formation. The results show that the fluoride-induced increased bone formation in osteoporotic patients is associated with an increased in vitro proliferative capacity of osteoblastic cells lining the trabecular bone surface, whereas parameters of osteoblast differentiation are not affected. The data also suggest that induction of a higher than normal bone cell proliferation is prerequisite for the stimulation of bone formation by fluoride.
New approaches for interpreting projected bone densitometry data
Authors: Professor Carter Dennis R. , Bouxsein Mary L. , Marcus Robert
Published: February 2010

Bone densitometry using dual-photon absorptiometry (DPA) or dual-energy x-ray absorptiometry (DXA) has become a standard method for assessing bone mineral content in the spine and other skeletal regions. A projected areal density, referred to as bone mineral density (BMD,g/cm2), is normally calculated to assess regional bone density and strength. We demonstrate that this measure can be misleading when used to compare bones of different sizes due to inherent biases caused by bone thickness differences. For example, assuming that volumetric bone density remains constant and bony linear dimensions are proportional to height, a 20% increase in height would result in a 20% increase in both the thickness and the BMD of any bone. We describe new analysis methods to reduce the confounding effect of bone size, and we introduce a parameter, bone mineral apparent density (BMAD, g/cm3), that better reflects bone apparent density. Using this parameter, we calculate a quantity that serves as an index of bone strength (IBS, g2/cm4) for whole vertebral bodies. These analyses were applied to lumbar spine (L2–4) DXA measurements in a population of women 17–40 years old and appear to offer advantages to conventional techniques.
Effect of swimming on prednisolone-induced osteoporosis in elderly rats
Authors: Dr. Swissa-Sivan A. , Statter M. , Brooks G.A. , Azevedo J. , Viguie C. , Azoury R. , Greenfield C. , Oman S. , Leichter I. , Zinker B.A. , Menczel J.
Published: February 2010

We investigated the possible ameliorating and preventive effect of swimming on prednisolone-induced osteoporosis in elderly rats. A total of 48 female Sabra strain rats were randomly assigned to the following groups and treatments: (1) control (C), (2) swimming (S), (3) prednisolone-treated (CP), and (4) swimming + prednisolone (SP). An additional 8 rats were sacrificed and examined at the onset of the study. Groups C and S were sham injected; groups CP and SP were injected with prednisolone (Ultracorten), 80 mg/kg three times per week for 10 weeks. Groups S and SP swam 1 h daily, 5 days per week for 10 weeks. SP rats swam simultaneously with prednisolone administration. At the end of the swimming period, in vivo bone mineral content (BMC) measurements were performed on rat vertebrae L4-5 by single-photon absorptiometry. Later, the humerus and femur were removed for the following measurements: morphometric, bone density (BD) by Compton scattering technique, bone ion content by atomic absorption, and hydration fraction by proton magnetic resonance (PMR). We found that the humeral BD of S rats was greater by 14%for group S over C and 3% greater for group SP over CP (P < 0.05). Vertebral BMC was higher by 15% in group S over C and 11% higher for group SP over CP (P < 0.05). Femoral calcium (mg/g dry bone) ion content was higher by 5% in group S over C and 8% in group SP over CP group (P < 0.05). The fraction of bound water was higher by 36% in group S over C and 8% in group SP over CP group (P < 0.05). The hydration properties of the femur were examined by PMR determining the longitudinal (T1) and transverse (T2) relaxation times, referring to the freedom of the hydrogen proton movement of water molecules within bone tissue. T2 relaxation time was found to be significantly greater in swimming rats compared to controls (27%) and 58% in group SP over CP. T1 relaxation time was found to be significantly higher (23%) in group C over S and 17% in group CP over SP (P < 0.05). No significant morphometric differences were found among the different treatment groups. These results support the notion that swimming partially prevents prednisolone-induced osteoporosis in elderly rats.
1,25-Dihydroxyvitamin D3 inhibits the passive transfer of cellular immunity by a myelin basic protein-specific T cell clone
Authors: Lemire Jacques M. , Adams John S.
Published: February 2010

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] and related analogs have been shown to exert immunoinhibitory effects on activated lymphocytes in vitro. However, the effects of the hormone on the mammalian immune response in vivo have not been well studied. To examine the possible immunoactions of 1,25-(OH)2D3 in vivo, we employed a murine model of experimental autoimmune encephalomyelitis (EAE). In this model, T helper lymphocyte clones developed from lines of lymphocytes reactive to myelin basic protein (MBP) confer MBP immunoreactivity and demyelinating central nervous system disease on syngeneic, naive recipients of the T cell clone. Similar to peripheral blood mononuclear cells incubated with mitogen, the T cell clone evaluated in this study expressed a high-affinity specific receptor for 1,25-(OH)2D3 (VDR; Kin = 0.03 nM) upon exposure to MBP. The MBP-stimulated clone elicited a ninefold enhancement of the local delayed hypersensitivity (DTH) response when as few as 0.5 × 105 cells of the T cell clone were injected into the foot pad of recipient mice. The DTH response in the recipient was completely blocked when the clone was preincubated with ≥ 10−8 M 1,25-(OH)2D3 before transfer; the half-maximal inhibitory concentration of hormone (EC50) was 5 × 10−9 M. These data indicate that exposure of antigen-reactive T helper lymphocytes to a VDR saturating concentration of 1,25-(OH)2D3 can dramatically lessen the expression of immunoreactivity in vivo.
Relationship between collagen synthesis and expression of the osteoblast phenotype in MC3T3-E1 cells
Authors: Franceschi Renny T. , Iyer Bhanumathi S.
Published: February 2010

The MC3T3-E1 mouse calvaria-derived cell line has been used to study the role of collagen synthesis in osteoblast differentiation. MC3T3-E1 cells, like several previously characterized osteoblast culture systems, expressed osteoblast markers and formed a mineralized extracellular matrix only after exposure to ascorbic acid. Mineralization was stimulated further by β-glycerol phosphate. Ultrastructural observations indicated that the extracellular matrix produced by ascorbic acid-treated cells was highly organized and contained well-banded collagen fibrils. Expression of osteoblast markers followed a clear temporal sequence. The earliest effects of ascorbic acid were to stimulate type I procollagen mRNA and collagen synthesis (24 h after ascorbate addition), followed by induction of alkaline phosphatase (48–72 h) and osteocalcin (96–144 h) mRNAs. Procollagen mRNA, which was expressed constitutively in the absence of ascorbate, increased only twofold after vitamin C addition. In contrast, alkaline phosphatase and osteocalcin mRNAs were undetectable in untreated cultures. Actions of ascorbic acid on osteoblast marker gene expression are mediated by increases in collagen synthesis and/or accumulation because (1) parallel dose-response relationships were obtained for ascorbic acid stimulation of collagen accumulation and alkaline phosphatase activity, and (2) the specific collagen synthesis inhibitors, 3,4-dehydroproline and cis-4-hydroxyproline, reversibly blocked ascorbic acid-dependent collagen synthesis and osteoblast marker gene expression.
Inhibition of bone resorption in vitro by human enkephalinase (EC 3.4.24.11), a neutral metalloendopeptidase
Authors: Dr. Ibbotson Kenneth J. , D'souza Sharyn M. , Deschodt-Lanckman Monique , Appelboom Thierry E.
Published: February 2010

Bone metabolism is regulated by a wide variety of both circulating and locally produced peptides. The activity of such agents must be regulated, and one potential regulating mechanism is the inactivation of these peptides by locally produced proteolytic enzymes. One candidate for such a class of enzymes is enkephalinase (EC 2.3.24.11), a membrane-bound neutral metalloendopeptidase that inhibits the activity of a range of biologically active peptides, including interleukin-1 (IL-1), a potent bone-resorbing agent. In this study, we examined the effects of human enkephalinase on bone resorption in cultures of fetal rat long bones. We found that partially purified and highly purified enkephalinase inhibited bone resorption stimulated by parathyroid hormone (PTH) and IL-1α. The effects on PTH-stimulated resorption were reversible, but enkephalinase did not inhibit prestimulated resorption. Enkephalinase also inhibited resorption induced by the nonpeptide stimulators 1,25-(OH)2D3, retinoic acid, and prostaglandin E2 (PGE2). In addition, preliminary studies confirmed a previous report of the presence of an enkephalinase-like activity in osteoblast-like osteosarcoma cells. These data are consistent with the hypothesis that proteolytic enzymes, such as enkephalinase, may play a role in the local regulation of bone resorption.
Developmental changes in calcium kinetics in children assessed using stable isotopes
Authors: Abrams Steven A. , Esteban Nora V. , Vieira Nancy E. , Sidbury James B. , Specker Bonny L. , Yergey Alfred L.
Published: February 2010

Total exchangeable calcium pool size (TEP) and bone calcium accretion rate (V0+) were measured using stable isotopes in healthy children and young adults. 42Ca or 46Ca was given intravenously to 10 children aged 10 months to 14 years and 3 women aged 23–33 years. Calcium kinetic parameters were determined using a two- or three-exponential curve of the resultant serum and urine tracer excesses. These data were compared with previously reported (radiotracer) kinetic studies of 21 children and 5 adults without known bone disease. Current results are comparable to those previously obtained, and the data from all studies were analyzed together. Total V0+ was significantly greater in children aged 3–16 years than in adults (2.8 ± 1.6 versus 0.7 ± 0.2 g/day, p < 0.01). Both TEP and V0+ were significantly correlated to age independently on variations in body weight (p < 0.01 for each). The ratio k0+ = V0+/TEP was greater in children than adults (0.36 ± 0.15 versus 0.12 ± 0.03 day−1, p < 0.001). These data demonstrate increased bone flow of calcium associated with increases in exchangeable calcium pools in children compared to adults. V0+ and TEP may be maximum in early adolescence, associated with peak rates of net calcium accretion. The use of stable isotopes permits the safe evaluation of calcium kinetics in patients of all ages.
Parathyroid hormone sensitizes long bones to the stimulation of bone resorption by 1,25-dihydroxyvitamin D3
Authors: Dr. van Leeuwen J. P. T. M. , Birkenhäger J. C. , Bos M. P. , van der Bemd G. J. C. M. , Herrmann-Erlee M. P. M. , Pols H. A. P.
Published: February 2010

In response to hypocalcemia the serum PTH level increases rapidly followed by a PTH-induced rise in 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] production. Therefore, bone is first exposed to increased PTH levels before increased 1,25-(OH)2D3 levels. In the present study the effect of pretreatment with PTH on 1,25-(OH)2D3-induced bone resorption was examined. Bone resorption was measured as release of prelabeled 45Ca during culture from 17-day-old fetal mice radii/ulnae and metatarsals. Radii/ulnae and metatarsals are characterized by differences in development. In radii/ulnae mature osteoclasts are present, whereas in metatarsals only different stages of preosteoclasts can be found. Preincubation for 24 h but not 4 h with PTH increases the stimulation of bone resorption by 1,25-(OH)2D3 in fetal radii/ulnae but not in metatarsals. Coincubation of PTH and 1,25-(OH)2D3 did not result in a significant change in bone resorption compared to 1,25-(OH)2D3 alone. The observed difference in the effect of pretreatment with PTH between radii/ulnae and metatarsals indicates that PTH does not stimulate the development of early osteoclast precursors but that a certain level of differentiation of the osteoclast precursor is required. Pretreatment with prostaglandin E2 resulted in an effect similar to that of PTH. Inhibition of prostaglandin synthesis by indomethacin prevented the potentiation of 1,25-(OH)2D3-induced bone resorption by pretreatment with PTH. Thus, the present study demonstrates that PTH sensitizes responses to 1,25-(OH)2D3. PTH must be present before 1,25-(OH)2D3 to observe a potentiation of 1,25-(OH)2D3-induced bone resorption. Local production of prostaglandins seems to be involved in this action of PTH. Taken together, these data suggest that besides increased renal production of 1,25-(OH)2D3 PTH also sensitizes bone for the 1,25-(OH)2D3-induced bone resorption.
Mineral crystals in calcified tissues: A comparative study by SAXS
Authors: Fratzl P. , Groschner M. , Vogl G. , Plenk H. , Eschberger J. , Fratzl-Zelman N. , Koller K. , Dr. Klaushofer K.
Published: February 2010

The shape, the typical orientation, and the average size of mineral crystals in different types of mineralized tissues were investigated by means of small-angle x-ray scattering (SAXS). To rule out eventual artifacts due to sample preparation, four different standard preparation techniques were used and a comparison showed that the SAXS results were identical for all four methods. In mineralized turkey leg tendon, a frequently used model system for bone, the crystals were found to be typically plate-like with a thickness of the order of 2 nm. This stands in contrast to the case of bone (calvaria, femur, and iliac crest) from mouse, rat, and dog, where mainly needle-like crystals were found. The thickness of these crystals ranged from 3 to 4 nm but was remarkably constant for different bones of a given animal. The preferred orientation of the needle-like crystals was along the main axis of the femur and within the surface of the calvaria (for mouse, rat, and dog). The mineral plates in turkey leg tendon were located inside the hole zone and oriented along the fibril axis. Finally, no periodic arrangement of the crystals inside the hole zone of the collagen could be found.
Integrins on rat osteoclasts: Characterization of two monoclonal antibodies (F4 and F11) to rat β3
Authors: Helfrich Miep H. , Nesbitt Stephen A. , Horton Michael A.
Published: February 2010

Two monoclonal antibodies, F4 and F11, were raised to newborn rat bone cell suspensions. These antibodies are shown by immunocytochemistry on tissue sections to recognize an antigen shared between osteoclasts, megakaryocytes, and platelets. Immunoprecipitation analysis of the antigen from C6 rat glial cells followed by SDS-PAGE showed a heterodimeric molecule with a characteristic integrin-like shift in apparent molecular mass upon reduction (137/78 kD nonreduced; 118/100 kD reduced); the low-molecular-mass band comigrates with the β3 subunit precipitated with polyclonal antihuman vitronectin receptor antiserum, and the high-molecular-mass band comigrates with the αv subunit precipitated with a polyclonal antiserum to a C-terminal amino acid sequence of human αv. Antibody F4 strongly cross-reacts with human cells and is shown in cross-blocking experiments and immunoprecipitation analysis with a human melanoma cell line DX3 to recognize a seemingly identical molecule as identified by anti-αvβ3 monoclonal antibody 23C6. Expression of F4 and F11 is reduced in platelets from a patient heterozygous for Glanzmann's thrombasthenia. Taken together, these results indicate that F4 and F11 recognize rat CD61, the integrin β3 chain, which, as was confirmed with polyclonal anti CD61 antisera, is highly expressed in rat osteoclasts. These antibodies may be useful tools in investigating the biochemical nature and biologic function of β3 integrins in rat osteoclasts. Additionally, because high expression of β3 in vivo is restricted to osteoclasts, megakaryocytes, and platelets, these antibodies may be used to help identify osteoclasts in tissue sections and bone cell suspensions. Apart from β3, rat osteoclasts were found to express high levels of αv and β1 integrins.
Derivation of osteoclasts from hematopoietic colony-forming cells in culture
Authors: Kerby J. A. , Hattersley G. , Collins D. A. , Chambers T. J.
Published: February 2010

The osteoclast is known to be derived from the hematopoietic stem cell, but its lineage remains controversial. There is evidence that osteoclastic differentiation is induced through a contact-dependent interaction between bone marrow stromal cells and hematopoietic precursors. To analyze osteoclastic lineage, colonies were generated in semisolid medium from mouse spleen cells in the presence of Wehi-conditioned medium, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or macrophage colony-stimulating factor (M-CSF) with or without erythropoietin (epo). After 5–8 days colonies were picked and phenotyped and incubated with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on bone slices or coverslips with bone marrow-derived cell lines (ts8 or ST2) that induce osteoclastic differentiation. Cells of osteoclastic phenotype [as judged by calcitonin receptor (CTR) expression or bone resorption] were observed only in multilineage colonies. The ability of cells that generate macrophage colonies (CFU-M) to generate osteoclasts was tested by incubating alveolar or peritoneal macrophages on ts8 or ST2 cells. Despite colony formation, no osteoclastic differentiation was detectable. Last, individual cells from blast cell colonies were incubated (1 cell per culture well) on ts8 or ST2 cells in the presence of 1,25-(OH)2D3 and epo (to expose the lineage potential of the plated cell). We found CTR-positive (CTRP) cells in 6 of 66 macrophage colonies, 7 of 12 granulocyte-macrophage (GM) colonies, and 49 of 50 colonies containing multiple lineages other than GM colonies. No single-lineage CTRP colonies were observed. Although most macrophage colonies did not contain CTRP, no CTRP were observed in colonies from which macrophages were absent. These results suggest that osteoclasts are derived from a multilineage precursor rather than from CFU-M.
Longitudinal study of bone metabolism after ethanol withdrawal in alcoholic patients
Authors: Pepersack Thierry , Dr. Fuss Michel , Otero Julio , Bergmann Pierre , Valsamis JosÉ , Corvilain Jacques
Published: February 2010

The pathogenesis of osteopenia in chronic alcoholism remains unclear, and many ethanol-related abnormalities have been advocated to explain bone loss. A direct inhibitory effect of ethanol on osteoblast function was suggested by in vivo and in vitro studies. We measured biochemical markers of bone turnover in 12 alcoholic men before and during a 2 week period of alcohol withdrawal, and we compared the results with those obtained in 15 nonalcoholic men. Our alcoholic patients presented with (1) decreased serum concentrations of bone gla protein (BGP), suggesting decreased bone formation; (2) increased urinary excretion of hydroxy-proline, suggesting increased bone resorption; (3) increased renal threshold of phosphate excretion without modification of serum PTH concentration, suggesting a direct effect of ethanol on the renal handling of phosphate. The rapid increase in serum BGP concentrations following ethanol withdrawal suggests that low serum BGP concentrations in alcoholics may result from a direct toxic effect of ethanol on osteoblast function and/or numbers.
Effects of phorbol myristate acetate on rat and chick osteoclasts
Authors: Murrills Richard J. , Stein Linda S. , Horbert Wendy R. , Dr. Dempster David W.
Published: February 2010

The role of protein kinase C in the regulation of osteoclast function is not known. We therefore compared the effect of phorbol myristate acetate (PMA), which activates protein kinase C, on the resorptive function, motility, and morphology of osteoclasts from rat and chick. PMA caused a significant reduction in resorption pit number in both species; rat osteoclasts were more sensitive, being significantly inhibited at doses of 10−9-10−6 M compared with 10−7-10−6 M for chick osteoclasts. The inactive analog PMA-α was without significant effect, and inhibition was not blocked by 10−6 M indomethacin. In time course experiments, inhibition at 24 h was similar to or greater than inhibition at 6 h, indicating a persistent or progressive effect on bone resorption. Removal of PMA after 6 h prompted partial recovery of bone-resorptive ability in chick osteoclasts but not rat, at least over a 48 h incubation. In time-lapse video studies of rat osteoclasts, 10−6 M PMA produced an immediate but transient cessation of motility and retraction of the cell margin into prominent filopodia. Motility resumed within 2.5 h after addition, but the osteoclasts remained partially contracted. Chick osteoclasts behaved similarly but showed no formation of filopodia at the cell periphery and a more rapid recovery of motility than rat osteoclasts; chick osteoclasts also underwent a transient vacuolation following PMA exposure, whereas rat osteoclasts did not. Despite differences in the sensitivity of rat and chick osteoclasts to PMA, these results suggest a fundamental role for protein kinase C in the inhibition of osteoclasts from both species.
Retention of etidronate in human, dog, and rat
Authors: Kasting Gerald B. , Francis Marion D.
Published: February 2010

The retention of radioactivity in human, rat, and dog following a single injected dose of radiolabeled etidronate disodium (EHDP) is shown to follow power-law decay curves with similar slopes for times up to 4, 60, and 80 days, respectively. During this period retention declines with time according to a weak inverse power of the time since dosing, with an exponent ranging from −0.05 (dog) to −0.09 (human and rat). Direct analyses of dog bones either 90 days after a single dose or 365 days after cessation of chronic dosing indicate a more rapid bone clearance of EHDP than predicted by the initial power law. Direct skeletal analysis also shows a more rapid loss of radioactivity in the rat between 60 and 365 days, indicative of either a second power law or a terminal exponential phase in the retention function occurring after 60 days. These data are used to estimate the minimum and maximum amounts of drug that would remain in the body following long-term treatment in humans. For the intermittent cyclic EHDP treatment (ICT) regimen for osteoporosis (repeated cycles of 14 daily doses of 400 mg orally followed by 76 days drug free), the projected retention of EHDP after 3 years of treatment is 25–50 times the daily absorbed dose. Thus, for a 60 kg woman with a daily absorbed dose of 12 mg, the retained mass of EHDP would be about 300–600 mg. The surface area available for diphosphonate adsorption combined with measured bone-remodeling rates in ICT-treated subjects dictates that this mass would be distributed into a sufficiently large volume of mineral such that the fractional occupancy of active bone surfaces is only a few percent. The predicted retained mass of diphosphonate is well below that known to inhibit mineralization. This analysis is consistent with the clinical histopathology of bone from ICT-treated subjects, which has indicated that no mineralization defect ensues from this treatment regimen with EHDP.
Specific binding of parathyroid hormone to living osteoclasts
Authors: Agarwala Neena , Gay Carol V.
Published: February 2010

We show that osteoclasts bind parathyroid hormone (PTH) in a manner that displays the properties of receptor-dependent hormone binding, that is, saturability, time dependence, temperature dependence, and hormone specificity. Osteoclasts were isolated from the endosteum of 2 to 3 week chick tibiae and maintained in culture for 4–6 days. Bovine PTH-(1–84) was biotinylated with N-hydroxysuccinimidobiotin. Biotinyl-PTH (btPTH, 10−5-10−11 M) was added to the cultured osteoclasts for 2–20 minutes. After rinsing away unbound btPTH, fluorescein isothiocyanate-labeled avidin (FITC-avidin) at a concentration of 66 μg/ml was applied. Receptor binding characteristics were assessed: (1) saturation occurred at around 10−6 M btPTH; (2) competition of excess unlabeled PTH was found, namely, a 10-fold excess abolished fluorescence; (3) specificity was shown by adding other polypeptide hormones (insulin, glucagon, and calcitonin) in 10- to 100-fold excess—no effect on PTH binding was observed; and (4) affinity of btPTH for its binding site was indicated by half-maximal binding ≅10−7 M for both osteoclasts and osteoblasts. Biotin (10−5 M) or FITC-avidin (66 μg/ml) alone did not cause fluorescence. The time course of btPTH on the cell exterior was short: at 2 and 5 minutes dots of fluorescence were randomly dispersed over the cell surface, by 10 minutes most of the fluorescence was clustered in one region of the membrane, and by 20 minutes most of the hormone was no longer present on the surface of the cells. This sequence of events and the finding that maintaining the cells at 4°C blocked the clustering process indicates that occupied receptors were rapidly internalized by endocytosis. Fibroblasts and osteoblasts were more intensely stained by btPTH-FITC-avidin than osteoclasts, and no internalization of label was observed over the time period studied. This study shows direct, short-term binding of PTH to osteoclasts through specific receptors. The binding of PTH to both osteoclasts and osteoblasts suggests dual control of bone resorption.
Prostaglandin e2 promotes osteoclast formation in murine hematopoietic cultures through an action on hematopoietic cells
Authors: Collins D.A. , Chambers T.J.
Published: February 2010

Osteoclastic differentiation is induced from hematopoietic cells in the presence of 1,25-(OH)2D3 by stromal cells that are present in bone but not in hematopoietic spleen. Recent evidence suggests that prostaglandins (PGs) are essential for this process. In this communication we describe experiments in which we have examined further the role of PGE2 in osteoclast formation. We found a marked reduction in basal, 1,25-(OH)2D3, and IL-3-induced production of calcitonin receptor (CTR)-positive cells and bone resorption by cyclooxygenase inhibitors, which was restored by PGE2 addition. Although some stromal cell types (ST2 cells) that support osteoclast formation from spleen cells produced PGs in response to 1,25-(OH)2D3, others (ts8 and calvarial cells) did not, either alone or in combination with spleen cells. On the other hand, both bone marrow and spleen cells produced amounts of PGE2 in response to 1,25-(OH)2D3 that were sufficient to account for osteoclast formation. Osteoclast-inductive ts8 cells were able to support osteoclast formation from spleen cells in the presence of 1,25-(OH)2D3 or PGE2 even if devitalized. Incubation of ts8 cells in these agents before devitalization did not avoid the requirement for the presence of PGE2 or 1,25-(OH)2D3 during subsequent incubation with spleen cells. Thus, hematopoietic cells produce sufficient PGE2 for osteoclast formation, and the PGE2 thus produced acts on hematopoietic precursors, which can be induced in the presence of PGE2 to express CTR and resorb bone on contact with osteoclast-inductive stromal cells. The ability of osteoclast-inductive cells to support osteoclast formation appears not to rest on their ability to produce, induce, or respond to PGE2.