bluuebunny/crossref_metadata_2025 huggingface dataset:
A new bisphosphonate, BM 21.0955, prevents bone loss associated with cessation of ovarian function in experimental dogs
Authors: Monier-Faugere Marie-Claude , Friedler Robert M. , Bauss Frieder , Malluche Hartmut H.
Published: February 2010

We previously found that bone loss occurs as soon as 1 month after ovariohysterectomy (OHX) in beagle dogs. Indirect evidence pointed to an early dramatic increase in bone resorption. To verify this hypothesis and evaluate the effects of a newly developed bisphosphonate, BM 21.0955 (Boehringer Mannheim), 36 beagle dogs were subjected to OHX and 12 dogs were sham operated (Sham). OHX dogs were divided into six groups (n = 6 each) and received subcutaneous injections of vehicle or BM 21.0955 at various doses (0.1, 0.3, 1, 10, and 100 μg/kg/day) for 1 month. Sham dogs were given vehicle (n = 6) or BM 21.0955 (1 μg/kg/day, n = 6). Iliac crest biopsies and blood drawings were done at baseline and at month 1. OHX dogs given vehicle exhibited a decrease in cancellous bone volume associated with an increase in erosion depth and a decrease in serum levels of 1,25-dihydroxyvitamin D. BM 21.0955 prevented the bone loss at a dose ≥ 1 μg/kg and the increase in erosion depth and the decrease in serum levels of 1,25-(OH)2D at a dose ≥ 0.3 μg/kg. No osteomalacia was observed at any dose of BM 21.0955. Bone turnover was reduced only when BM 21.0955 was administered at doses of 10 or 100 μg/kg. There were no changes in body weight or serum levels of calcium, phosphorus, creatinine, parathyroid hormone, or osteocalcin in all groups. The increase in erosion depth in OHX dogs given vehicle proves that the early rapid bone loss after cessation of ovarian function is related to an increase in osteoclastic activity. The antiosteoclastic activity of BM 21.0955 at a dose ≥ 1 μg/kg prevents this increase and preserves bone volume. The absence of any signs of osteomalacia at any dose confers a relatively wide therapeutic margin to BM 21.0955. BM 21.0955 at a dose ≥ 10 μg/kg also acts as an inhibitor of bone turnover. This is not observed at a dose of 1 μg/kg, at least after 1 month of administration.
Differential effects of fluoride during initiation and progression of mineralization of osteoid nodules formed in vitro
Authors: Dr. Bellows C.G. , Aubin J.E. , Heersche J.N.M.
Published: February 2010

Osteoid nodules form in cultures of fetal rat calvarial (RC) cells grown in medium containing 10% FBS and 50 μg/ml of ascorbic acid. When 10 mM β-glycerophosphate (β-GP) is added, osteoid nodules mineralize in two phases: an initiation phase, which is dependent upon alkaline phosphatase activity for conversion of β-GP to Pi, and a progression phase that proceeds independently of alkaline phosphatase activity and does not require exogenous phosphate. We have now used this system to investigate the effects of fluoride (F−) on mineralization. In cultures in which osteoid was formed and mineralization initiated in the presence of F−, a dose-dependent inhibition of the initiation of mineralization occurred over a concentration range of 25–500 μM F− (p < 0.001 in all cases). The initiation of mineralization was not inhibited if F− was removed from the cultures at the time when mineralization was initiated with β-GP. In osteoid nodules grown in the absence of F−, addition of F− resulted in a dose-dependent inhibition of the initiation of mineralization, with significant decreases in 45Ca uptake occurring at F− concentrations of 3 μM (p < 0.01) and higher. However, if F− was added to cultures after mineralization was initiated in the absence of F−, a stimulation of 45Ca uptake was observed at F− concentrations of 250 μM and above (p < 0.001). F− (1–1000 μM) did not affect the conversion of β-GP to Pi or alkaline phosphatase activity in the cultures. The data show that mineralization of osteoid formed in the presence of F− is unaffected when F− is removed before initiation, that F− inhibits the initiation phase of mineralization at concentrations as low as 3 μM, and that F− stimulates mineralization during the progression phase. These data agree with clinical observations of hypermineralization of existing bone and hypomineralization of newly formed osteoid in association with F− administration.
Cell signaling and estrogens in female rat osteoblasts: A possible involvement of unconventional nonnuclear receptors
Authors: Dr. Lieberherr Michèle , Grosse Brigitte , Kachkache Moustapha , Balsan Sonia
Published: February 2010

Estrogen deficiency is associated with bone loss, and estrogen replacement is an effective treatment of this osteoporotic process. This study examines the early (5–120 s) effects of 17β-estradiol on the intracellular calcium and phospholipid metabolism in confluent female rat osteoblasts. The cytosolic free Ca2+ concentration ([Ca2+]i) was determined using fura-2/AM as Ca2+ probe. Cells were labeled with myo-[2-3H]inositol or [14C]arachidonic acid for inositol or lipid determination. Inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) production were determined by either mass measurement or anion-exchange chromatography or by thin-layer chromatography, respectively. 17β-Estradiol (1 pM to 1 nM) increased [Ca2+]i in a biphasic manner within 10 s via Ca2+ influx from the extracellular milieu, as shown by the effects of the calcium chelator EGTA and the Ca2+ channel blockers nifedipine and verapamil, and via Ca2+ mobilization from the endoplasmic reticulum (ER), as shown by the effects of thapsigargin. 17β-Estradiol (1 pM to 1 nM) induced a biphasic and concomitant increase in IP3 and DAG formation. Estradiol immobilized on bovine serum albumin (BSA) [E-(O-carboxymethyl)oxime BSA] and its derivative (O-carboxymethyl)oxime rapidly increased ([Ca2+]i, IP3, and DAG and were full agonists, although they were less potent than the free estradiol. They had the same action time course and acted via Ca2+ influx and Ca2+ mobilization from ER. Tamoxifen, a potent inhibitor of genomic steroid responses, did not block the rapid increase in Ca2+, IP3, and DAG induced by estradiol. Finally, inhibitor of phospholipase C (neomycin) and pertussis toxin abolished the effects of 17β-estradiol on IP3 and DAG formation. These results suggest that female rat osteoblasts bear non-genomic unconventional cell surface receptors for estradiol, belonging to the class of the membrane receptors coupled to a phospholipase C via a pertussis toxin-sensitive G protein.
Human 25-hydroxyvitamin D 24-hydroxylase cytochrome P450 subunit maps to a different chromosomal location than that of pseudovitamin D-deficient rickets
Authors: Labuda Malgorzata , Lemieux Nicole , Tihy Fréderique , Prinster Chiara , Dr. Glorieux Francis H.
Published: February 2010

We have cloned part of the human 25-OHD 24-hydroxylase cytochrome P450 (P450cc24) cDNA. The characterized sequence consists of 776 bp of the coding and 720 bp of the 3′-untranslated region interrupted by an intron. In the coding region we found 79.8% similarity in DNA and 87.5% in deduced amino acid sequences between human and rat, with no similarity in the 3-untranslated region. By Southern blot hybridization of DNA from human-hamster somatic cell hybrids and by in situ immunofluorescence hybridization, we mapped P450cc24 to human chromosome 20q13.1. This location of P450cc24 is different from that of pseudovitamin D-deficient rickets (PDDR), previously assigned to chromosome 12q14 by linkage analysis, thus excluding it as a target of the PDDR mutation. Since it is likely that PDDR is caused by a mutation in the 25-OHD 1α-hydroxylase P450 subunit (P450cc1α) our results do not support the hypothesis that the two cytochromes are encoded by a single gene.
Relationship between quadriceps strength and bone mineral density of the proximal tibia and distal forearm in women
Authors: Dr. Madsen Ole Rintek , Schaadt Ole , Bliddal Henning , Egsmose Charlotte , Sylvest Jesper
Published: February 2010

It is well known that there is a relationship between muscle strength and bone density, but it is uncertain whether this relationship is site specific. The aim of this study was to assess the relationship of quadriceps strength to site-specific bone mineral density (BMD) of the tibia and to BMD of the forearm. In 66 healthy women, aged 21–78 years, BMD was measured in the proximal tibia and the distal forearm by dual-photon absorptiometry. Isometric and isokinetic strength of the quadriceps was measured using an isokinetic dynamometer (Cybex II). Highly significant correlations between BMD of the proximal tibia and quadriceps strength were found (RS ranging from 0.79 to 0.84, p < 0.0001). Also, BMD of the distal forearm was correlated with quadriceps strength (RS ranging from 0.59 to 0.62, p < 0.0001). In a stepwise multiple regression analysis, quadriceps strength was a better predictor of tibial BMD than age, body height, or weight. However, age, height, and weight were more predictive of forearm BMD than quadriceps strength. When studying the pre- and postmenopausal women separately, quadriceps strength was correlated with BMD of the proximal tibia but not to forearm BMD. In conclusion, the study provides support for a site-specific relationship between muscle and bone.
Effect of 1,25,28-trihydroxyvitamin D2 and 1,24,25-trihydroxyvitamin D3 on intestinal calbindin-D9K mRNA and protein: Is there a correlation with intestinal calcium transport?
Authors: Wang Y.-Z. , Li H. , Bruns M.E. , Uskokovic M. , Truitt G.A. , Horst R. , Reinhardt T. , Dr. Christakos Sylvia
Published: February 2010

Although analogs and metabolites of vitamin D have been tested for their calciotropic activity, very little information has been available concerning the effects of these compounds on gene expression. In this study one analog of vitamin D, 1,25,28-trihydroxyvitamin D2 [1,25,28-(OH)3D2], and one metabolite, 1,24,25-trihydroxyvitamin D3 [1,24,25-(OH)3D3], were tested for their effect on intestinal calbindin-D9K mRNA and protein as well as for their effect on intestinal calcium absorption and bone calcium mobilization. These compounds were also evaluated for their ability to compete for rat intestinal 1,25-(OH)2D3 receptor sites and to induce differentiation of human leukemia (HL-60) cells as indicated by reduction of nitro blue tetrazolium. In vivo studies involved intrajugular injection of 12.5 ng 1,25-(OH)2D3 or test compound to vitamin D-deficient rats and sacrifice after 18 h. 1,25,28-Trihydroxyvitamin D2 had no effect on intestinal calcium absorption, bone calcium mobilization, or intestinal calbindin-D9K protein and mRNA. Competitive binding to 1,25-(OH)2D3 receptors was 0.8% of that observed using 1,25-(OH)2D3. However, 20- and 40-fold higher doses of 1,25,28-(OH)3D2 (250 and 500 ng) resulted in significant inductions in calbindin-D9K protein and mRNA (3.5 to 7.4-fold), although doses as high as 800 ng were found to have no effect on intestinal calcium absorption or bone calcium mobilization. 1,25,28-Trihydroxyvitamin D2, although lacking in calciotropic activity, was found to induce differentiation of HL-60 cells at high concentrations [ED50 = 15 × 10−8 M compared to ED50 = 2.5 × 10−8 M for 1,25-(OH)2D3]. 1,24,25-Trihydroxyvitamin D3 was 93% as active as 1,25-(OH)2D3 in stimulating intestinal calcium transport but was relatively inactive in stimulating bone calcium mobilization. Competitive binding to the 1,25-(OH)2D3 receptor was 8% of that observed using 1,25-(OH)2D3. Although 1,24,25-(OH)3D3 was 93% as active as 1,25-(OH)2D3 in stimulating intestinal calcium absorption, this compound was found to be 50% as active as 1,25-(OH)2D3 in stimulating calbindin-D9K protein and mRNA. The lack of a direct correlation between calbindin protein and mRNA and intestinal calcium transport after 1,24,25-(OH)3D3 administration or after administration of high doses of 1,25,28-(OH)3D2 suggests that factors in addition to calbindin are involved, at least in part, in vitamin D-regulated intestinal calcium transport.
Parathyroid hormone-related peptide may increase mammary blood flow
Authors: Davicco Marie-Jeanne , Rouffet Josette , Durand Denis , Lefaivre Jacques , Barlet Jean-Pierre
Published: February 2010

Amino-terminal fragments of PTHrP were previously shown to increase regional blood flow in laboratory animals. Since PTHrP is produced in the lactating mammary gland and associated nutrient vessels, we examined the effects of peptide fragments of PTHrP on the hemodynamics of the mammary gland of dried sheep. The left arterial mammary blood flow measured using ultrasonic flow probes in four dried Lacaune ewes was 233 ± 11 ml/minute. It was significantly increased when synthetic human PTHrP-(1–34) or (1–86) fragments were injected into the mammary artery. The effect was dose dependent for PTHrP-(1–34), varying between 0.0075 and 0.3 nmol/kg body weight. PTHrP-(140–173) fragment lacked any vasorelaxant activity. Synthetic human endothelin (ET1) decreased arterial blood flow in a dose-dependent manner. This decrease was inhibited by PTHrP-(1–34), and this inhibition was PTHrP dose related. When ET1 (10 pmol/kg body weight) was injected together with PTHrP-(1–86) (100 pmol/kg body weight), only a significant increase in mammary blood flow was observed. Thus, PTHrP produced by the lactating mammary gland may be involved in the regulation of mammary blood flow.
Journal of Bone and Mineral Research
Authors: Raisz Lawrence G.
Published: February 2010

In an effort to define the major unanswered questions in bone cell biology and suggest new approaches to answering these questions, I have outlined the bone remodeling cycle and briefly described the major local and systemic factors that regulate bone cell function. These factors include calcium-regulating and systemic hormones as well as locally produced prostaglandins, cytokines, and growth factors. To understand the individual roles of this large number of regulators, it will be necessary to develop new approaches to measure their production and activity in bone under physiologic and pathologic conditions. Quantitative methods in molecular and cellular biology have been developed that should make this identification possible.
Journal of Bone and Mineral Research
Authors: Christiansen Claus
Published: February 2010

Osteoporosis is characterized by decreased bone mass and increased susceptibility to fractures. The clinical consequences of osteoporosis are fracture, most commonly seen at the wrist, the spine, and the hip. The prevalence of osteoporotic fracture is extremely high with 30%–40% of 70-year-old women having had at least one osteoporotic fracture. The prevalence in men is lower but may constitute a larger problem than expected. Osteoporotic fracture is, therefore, a significant cause of morbidity and mortality and represents a major problem of health care. Bone is the site of substantial metabolic exchanges, with bone resorption and formation continuing throughout life. The turnover processes are carried out by osteoclasts and osteoblasts in specialized cellular units. After cessation of growth, the skeleton probably consolidates to reach the peak bone mass at the age of 35–40 years. The relatively slow subsequent age-related bone loss occurs in both men and women, but women are additionally exposed to accelerated bone losses after the menopause. Estrogen deficiency is the dominating pathogenetic factor for osteoporosis in women. The role of disturbances in the calcium-regulating hormones and vitamin D deficiency is less sure. The pathophysiologic mechanisms of osteoporosis in men are not well investigated. Those who are endowed with a low peak bone mass and/or are exposed to a large bone loss are at particular risk of developing symptomatic osteoporosis. Several risk factors have been recognized but are insufficiently sensitive and specific to predict an individual's overall risk of developing osteoporosis.
Journal of Bone and Mineral Research
Authors: Prockop Darwin J. , Colige Alain , Helminen Heikki , Khillan J.S. , Pereira Ruth , Vandenberg Philipp
Published: February 2010

Work by a large number of investigators over the last decade has established that over 90% of patients with osteogenesis imperfecta have mutations in one of the two genes for type I procollagen, that most unrelated probands have different mutations in the genes, and that the mutations found in most of the serious variants of the disease cause synthesis of abnormal proα chains of the protein. The results have demonstrated that synthesis of structurally abnormal but partially functional proα chains can interfere with folding of the central region of the protein into a triple-helical conformation, prevent processing of the N-terminal propeptides of procollagen, or produce subtle alterations in conformation that interfere with the self-assembly of the protein into collagen fibrils. One of the unsolved mysteries about the disease is why some mutations produce severe phenotypes, whereas very similar mutations produce mild phenotypes. Recent studies in transgenic mice suggest that nongenetic factors, such as stochastic events during development, may determine the severity of the disease phenotype produced by a specific mutation. Also, recent results raised the possibility that strategies of antisense gene therapy may be effective in treating the disease some time in the future. Specific inhibition of expression of a mutated collagen gene has been obtained with antisense oligonucleotides in cell culture experiments. However, there is no means of selective delivery of antisense oligonucleotides to the appropriate tissues.
Journal of Bone and Mineral Research
Authors: Delmas Pierre D.
Published: February 2010

The noninvasive assessment of bone turnover has received increasing attention over the past few years because of the need for sensitive markers in the clinical investigation of osteoporosis. Markers of bone formation include the serum measurement of total and bone-specific alkaline phosphatase, osteocalcin, and type I collagen extension peptides. Assessment of bone resorption can be achieved with measurement of fasting urinary calcium and hydroxyproline, urinary hydroxylysine glycosides, urinary excretion of the pyridinium cross-links (pyridinoline and deoxypyridinoline), and plasma tartrate-resistant acid phosphatase activity. Several studies performed in a variety of metabolic bone diseases have shown these markers to be of unequal sensitivity and specificity. In addition, some of them are not fully characterized. For assessment of the level of bone turnover in women with vertebral osteoporosis, serum osteocalcin and urinary pyridinoline appear to be the most sensitive markers so far. Programs combining bone mass measurement and assessment of bone turnover by several markers in women at the time of menopause are being developed in an attempt to improve the assessment of the risk for osteoporosis. Efforts are being made to develop more convenient assays and to identify other markers of bone turnover. A battery of various specific markers is likely to improve the assessment of the complex and subtle anormalities of bone metabolism that characterize metabolic bone diseases, especially the various aspects of osteoporosis.
Journal of Bone and Mineral Research
Authors: Boyan Barbara D. , Schwartz Zvi
Published: February 2010

Study of oral tissues to understand the mechanisms of osteoporosis and oral bone loss includes histologic, biochemical, and molecular assessments of the tissue itself, as well as in vivo analysis of the biology of resident cells. Tissue sampling is limited by the nature of the defect and the use of appropriate controls (contralateral site vs same site, different subjects vs repeated measures of the same sites). Experimental parameters may include histomorphometrics, histochemistry, immunohistochemistry, and in situ hybridization. Molecular and biochemical technology also can be used to study the tissue in vivo. The presence of mineral is a confounding variable. To understand the underlying mechanisms of oral bone loss, cell culture is a powerful tool. The location in the oral cavity, the type of tissue (periosteum/cortical bone/trabecular bone), and the presence of pathology (periodontal disease) affect the biology of the cultured cells. Enzymatic release of cells from their extracellular matrix yields heterogeneous cell populations. Migratory cells from explant cultures are more homogeneous but less differentiated. Fibroblastic and bacterial contamination may be problems. Although cell culture data must be considered in the context of the intact tissue, the potential exists for developing bone cell function tests with diagnostic use in the treatment of bone disease.
Journal of Bone and Mineral Research
Authors: Rubin Clinton T. , Donahue Henry J. , Rubin Janet E. , McLeod Kenneth J.
Published: February 2010

The discovery of piezoelectric potentials in loaded bone was instrumental in developing a plausible mechanism by which functional activity could intrinsically influence the tissue's cellular environment and thus affect skeletal mass and morphology. Using an in vivo model of osteopenia, we have demonstrated that the bone resorption that normally parallels disuse can be prevented or even reversed by the exogenous induction of electric fields. Importantly, the manner of the response (i.e., formation, turnover, resorption) is exceedingly sensitive to subtle changes in electric field parameters. Fields below 10 μV/em, when induced at frequencies between 50 and 150 Hz for 1 h/day, were sufficient to maintain bone mass even in the absence of function. Reducing the frequency to 15 Hz made the field extremely osteogenic. Indeed, this frequency-specific sinusoidal field initiated more new bone formation than a more complex pulsed electromagnetic field (PEMF), though inducing only 0.1% of the electrical energy of the PEMF. The frequencies and field intensities most effective in the exogenous stimulation of bone formation are similar to those produced by normal functional activity. This lends strong support to the hypothesis that endogenous electric fields serve as a critical regulatory factor in both bone modeling and remodeling processes. Delineation of the field parameters most effective in retaining or promoting bone mass will accelerate the development of electricity as a unique and site-specific prophylaxis for osteopenia. Because fields of these frequencies and intensities are indigenous to bone tissue, it further suggests that such exogenous treament can promote bone quantity and quality with minimal risk or consequence.
Journal of Bone and Mineral Research
Authors: Legeros Racquel Z. , Craig Ronald G.
Published: February 2010

Osteointegration was defined as a “direct structural and functional connection between ordered living bone and the surface of a load-carrying implant.”(25) Although osteointegration was meant originally to describe a biologic fixation of the titanium dental implants, it is now used to describe the attachment of other materials used for dental and orthopedic applications as well. Analyses of material-bone interface showed that osteointegrated implants can have an intervening fibrous layer or direct bone apposition characterized by bone-bonding(62) depending on the composition and surface properties of the biomaterial. This article reviews biologic (host tissue properties and response), biomechanical, and biomaterial factors affecting osteointegration. Biologic factors include the quality of bone. Biomaterial factors include the effect of material composition on the bone-material interface. Suggested areas for future research include determining the correlation between oral bone status and osteoporosis, the effect of gender, age, and endocrine status (e.g., osteoporosis) on implant success or failure, the effect of calcium phosphate coating composition and crystallinity on in vivo performance of implants, the factors contributing to accelerated osteointegration, and development of osteoinductive implants.
Automated three-dimensional characterization of osteoclastic resorption lacunae by stereoscopic scanning electron microscopy
Authors: Fuller K. , Thong J.T.L. , Breton B.C. , Chambers T.J.
Published: February 2010

The use of stereoscopic scanning electron microscopy to analyze quantitatively the topography of excavations made by osteoclasts in slices of devitalized cortical bone was evaluated. Using this innovative technique, the need mechanically to tilt the specimen stage to gather three-dimensional information is obviated by instead tilting the electron beam both to produce real-time stereo pairs and to gather measurement data. Based on the comparison of two images of microscopic areas, cross-correlation is used to determine the image shift between the features in the stereo pair. This information is then used dynamically to correct the height of the tilt axis and lens focus in a feedback loop, generating a quantitative measurement of height difference. With this approach, relative heights of individual points, line profiles, area topography maps, and three-dimensional reconstructions of excavations were obtained rapidly and at high resolution. This approach combines the advantage in topographic data acquisition of confocal microscopy with the advantages of the increased resolution and focal depth of scanning electron microscopy (SEM). The technique should facilitate not only the topographic analysis of osteoclastic excavations in bone slices at high resolution but also the three-dimensional analysis of the structure of bone tissue.
Histochemical and autoradiographic studies on elcatonin internalization and intracellular movement in osteoclasts
Authors: Ikegame Mika , Ejiri Sadakazu , Professor Ozawa Hidehiro
Published: February 2010

The binding sites and chronologic localization of elcatonin (eCT) in osteoclasts were examined by autoradiography using [125I]elcatonin (125I-eCT). In addition to the structural changes induced by calcitonin (CT) reported so far, changes were also observed in the structure of Golgi apparatus. These changes continued until 48–72 h after incubation with eCT. Developed silver grains of 125I-eCT were localized into multinucleated osteoclasts and mononuclear cells that were ultrastructurally defined as “preosteoclasts.” The silver grains located on plasma membranes of those cells and were then internalized; they accumulated, especially in the Golgi apparatus, and remained for 48–72 h. A few silver grains were also detected in lysosomes and small vesicles. The decrease in the number of silver grains in the Golgi apparatus accompanied the recovery of osteoclast structures – Golgi apparatus and then ruffled borders. These findings suggest that (1) CT especially inhibits the sorting function of Golgi apparatus in osteoclasts, resulting in prolonged retention of CT in this organelle. (2) The CT in Golgi apparatus may keep its activity and cause the prolonged effect of CT on osteoclast activity.
Calcitriol corrects deficient calcitonin secretion in the vitamin D-deficient elderly
Authors: Quesada J.M. , Mateo A. , Jans I. , Rodriguez M. , Bouillon R.
Published: February 2010

The thyroid calcitonin-producing C cells possess vitamin D receptors and synthesize the vitamin D-dependent calbindin D28K. The present study evaluates the possible direct or indirect influence of vitamin D on calcitonin secretion in the elderly. Serum calcitonin was measured before and after a short calcium infusion (1.5 mg/kg over 10 minutes) in nine normal young adults (30 ± 4 years, mean ± SEM) and eight elderly subjects (78 ± 4 years). The test was repeated 48 h after the last of three intravenous injections of calcitriol (2 μg) given every other day. Basal serum calcium did not change, but basal calcitonin of the elderly increased from 7 ± 1 to 10 ± 1 pg/ml (p < 0.06), similar to basal values in young adults (11 ± 1 pg/ml). The increase in calcitonin after calcium infusion increased from 8 ± 1 to 14 ± 1 pg/ml (p < 0.001) after calcitriol treatment and approached the increase in young adults (18 ± 3 pg/ml). These data demonstrate that calcitriol can improve and nearly normalize the impaired calcitonin secretion of the mildly vitamin D-deficient elderly subjects without changes in serum calcium, whereas the inverse situation is observed for parathyroid hormone.
Influence of age on cyclosporin A-induced alterations in bone mineral metabolism in the rat in vivo
Authors: Katz Ian , Li Mei , Joffe Ian , Stein Barry , Jacobs Timothy , Liang Xiao Guang , Ke Hua Zhu , Jee Webster , Dr. Epstein Sol
Published: February 2010

Cyclosporin A (CsA) administered to actively growing young rats produces a high-turnover osteopenia. We investigated and compared the effect of CsA on the bone mineral metabolism in young rats with that of older rats, which have a lower rate of bone turnover. A group of 24 young (9 weeks) and 24 older (9 months) male Sprague-Dawley rats were orally administered 15 mg/kg of CsA or placebo daily for 24 days. Rats were weighed and serum assayed serially for bone gla protein (BGP), parathyroid hormone, ionized calcium, blood urea nitrogen, creatinine, and 1,25-dihydroxyvitamin D [1,25-(OH)2D]. After sacrifice, histomorphometric analysis was performed on undecalcified proximal tibial metaphysis with double-fluorescent labeling. Serum BGP levels were significantly elevated in both young and older rats administered CsA, and 1,25-(OH)2D levels were significantly elevated in CsA-treated young rats more than in older rats. Body weight was significantly reduced in CsA-treated older rats. There were mild but significant alterations in renal function in both groups receiving CsA. In the most comprehensive examination to date of the effects of CsA on bone histomorphometry, both young (-44%) and older rats (-20%) lost significant amounts of trabecular bone compared to their respective controls. Bone loss in young rats was mainly due to a reduced number of trabeculae; older rats lost mainly trabecular thickness. Microanatomic nodal studies were consistent with these results. These data demonstrate that although cancellous bone loss induced by CsA is more marked in young rats, older rats with slower bone turnover are also at risk.
Interleukin-6, IL-6 receptor, and IL-6 nuclear factor gene expression in paget's disease
Authors: Dr. Hoyland Judith A. , Freemont A.J. , Sharpe P.T.
Published: February 2010

The cytokine interleukin-6 (IL-6) is considered an important regulator of bone cell function and may play a central role in bone disease states characterized by increased bone remodeling, such as Paget's disease. Indeed, recent in vitro data suggest that IL-6 may be an autocrine/paracrine factor for pagetic osteoclasts. However, its expression and role in vivo are not known. Using in situ hybridization we investigated the spatial localization of expression of IL-6, IL-6 receptor (IL-6R), and the transcription factor (NF-IL-6) in pagetic bone. Our results show that osteoblasts in the normal remodeling bone of osteoarthritis (controls) and in Paget's disease express IL-6, IL-6R, and NF-IL-6 genes with higher levels of IL-6 and IL-6R mRNA in pagetic bone. Osteoclasts in both osteoarthritic and pagetic bone express IL-6R mRNA and NF-IL-6, but only pagetic osteoclasts expressed IL-6, suggesting that in Paget's disease IL-6 can act as an autocrine factor on osteoclasts. These results provide evidence for a major role of the IL-6 regulatory pathway in the phenotype of the pagetic osteoclasts and lead us to suggest a model linking possible paramyxovirus infection and IL-6 regulation in the pagetic osteoclast.
Long-term elevation of 1,25-dihydroxyvitamin D after short-term intravenous administration of pamidronate (aminohydroxypropylidene bisphosphonate, APD) in paget's disease of bone
Authors: Devlin Rowena D. , Retallack Robert W. , Fenton Anna J. , Grill Vivian , Gutteridge Donald H. , Kent Neil G. , Prince Richard L. , Worth Graeme K.
Published: February 2010

We report the prolonged biochemical changes that occurred in patients with Paget's disease when treated for 2–10 days with pamidronate disodium (3-amino-1-hydroxypropylidine-1,1-bisphosphonate, APD), by IV administration and observed for 6 months following therapy. In all 24 patients studied, bone resorption (measured by urinary hydroxyproline/creatinine ratio, OHP/Cr) fell sharply on treatment, from 0.12 ± 0.02 (mean ± SEM; above reference limits) to 0.04 ± 0.008 (reference range 0.006–0.027 for females, 0.005–0.020 for males), remaining at this level for 6 months after therapy. A fall in serum ionized calcium (Ca2+) to just below the reference limits with treatment (1.11 ± 0.02 mM; reference range 1.14–1.18 mM), followed by a rapid return to normal levels (1.14 ± 0.02 mM, mean ± SEM) within 8 days of treatment, was presumably due to the cessation of release of calcium from bone. This was followed by secondary hyperparathyroidism and a rise in serum 1,25-dihydroxyvitamin D [1,25-(OH)2D]. The hormonal responses, however, were profound. Serum immunoreactive PTH (iPTH) rose to twice pretreatment values (86 ± 11 pM, mean ± SEM; reference range for iPTH, >50 years, <50 pM; <50 years, <40 pM), returning to normal 4–8 weeks after therapy. Serum 1,25-(OH)2D levels rose to three times pretreatment values (300 ± 20 pM, mean ± SEM; reference range 50–150 pM), remaining above reference limits 4–8 weeks after therapy (188 ± 15 pM, mean ± SEM) and returning to normal values only after 12 weeks. The magnitude of the hormonal responses leads to speculation that factors other than serum calcium are involved in affecting these levels. Serum 24,25-dihydroxyvitamin D [24,25-(OH)2D] levels were below the mean of the reference range before treatment (3.93 ± 0.5 nM, mean ± SEM) and fell significantly with APD therapy (2.44 ± 0.25 nM, mean ± SEM), returning to pretreatment values only 12 weeks later (4.14 ± 0.35 nM). Serum 25-hydroxyvitamin D (25-OHD) did not change with treatment. We previously reported an increase in serum 1,25-(OH)2D levels in patients with Paget's disease treated with calcitonin (CT) or etidronate (EHDP). We now find a more pronounced and prolonged change in serum 1,25-(OH)2D with short-term IV APD treatment, which could indicate that with this bisphosphonate there is a long-term increase in gut calcium absorption. Also, 1,25-(OH)2D may play a role in the healing of pagetic lesions whether the treatment is CT, EHDP, or APD.
Transforming growth factor β increases ecto-nucleoside triphosphate pyrophosphatase activity of human bone-derived cells
Authors: Oyajobi Babatunde O. , Caswell Alison M. , Russell R. Graham G.
Published: February 2010

Inorganic pyrophosphate (PPi) may be involved in the regulation of mineralization. The cell surface enzyme, ecto-NTP pyrophosphatase, could be a major source of extracellular PPi in bone, and agents that influence its activity in osteoblasts may modulate bone mineralization. We studied the effects of serum on the ecto-NTP pyrophosphatase activity of cultured human osteoblast-like cells. Enzyme activity was lowered when the concentration of fetal calf serum (FCS) was reduced from 10 to 2.5% (vol/vol) for 48 h, and a further decrease in activity was observed after 96 h. Relative to enzyme activity in cells cultured in serum-free medium for 96 h, adult human platelet-poor plasma (HPPP; 2.5–10% vol/vol) induced a small increase, similar concentrations of adult human serum (HS) induced much larger increases, and charcoal-depleted FCS was ineffective. In an attempt to identify the factor(s) present in serum that influence ecto-NTP pyrophosphatase activity, we examined transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF). PDGFs AA, AB, and BB (0.1–10 ng/ml) were ineffective, but both TGF-β1 and TGF-β2 increased enzyme activity. The increase was dose dependent between 0.001 and 10 ng/ml, was enhanced in the presence of 2% vol/vol FCS, and was not potentiated by PDGF or by 1,25-(OH)2D3. Furthermore, the increase was independent of cell density and was blocked by inhibitors of protein and RNA synthesis. Ecto-NTP pyrophosphatase of subject-matched human dermal fibroblasts was unaffected by TGF-β (10 ng/ml), suggesting that modulation of activity by the growth factor may be tissue specific. Alkaline phosphatase (ALP) probably serves to hydrolyze extracellular PPi in bone. In contrast to effects on NTP pyrophosphatase activity in osteoblast-like cells, TGF-β1 and TGF-β2 (0.001–10 ng/ml) decreased ALP activity dose dependently after 72 h. By inducing opposing changes in ecto-NTP pyrophosphatase and ALP activities, TGF-β may increase extracellular PPi concentrations in osseous tissues and consequently modulate bone mineral properties in vivo.
Effect of the bisphosphonate risedronate on bone metastases in a rat mammary adenocarcinoma model system
Authors: Hall D. Greg , Stoica George
Published: February 2010

Risedronate (NE-58095) is a third-generation bisphosphonate with very potent antiresorptive activity but few toxic effects. The purpose of this work was to evaluate the effect of risedronate treatment on bone metastases produced in a rat breast cancer model. Berlin Druckrey IV rats inoculated with ENU1564 mammary adenocarcinoma cells were treated daily with risedronate or a saline placebo. Survival times, dictated by extraskeletal metastases (lung, heart, and brain), were not affected by risedronate treatment. Risedronatetreated animals had skeletal changes associated with decreased remodeling of bones undergoing endochondral ossification, most prominently affecting the appendicular skeleton. Despite the skeletal alterations induced by the treatment, the distribution of bone metastases throughout the surveyed skeletal sites was similar for treated and untreated animals. Bone metastases were enumerated in histologic sections of distal femur, spine, and skull. Tumor size was estimated from area measurements obtained from histologic lesions in distal femoral metaphyses and vertebral bodies. A greater number of treated rats had no bone metastases in any of the examined sections (30 versus 16.1% of untreated rats). Multiple bone metastases were observed less frequently in treated rats (33.3 versus 71% of untreated rats). Treated rats had fewer observed bone metastases in each examined site than untreated rats (p ≤ 0.025). Mean tumor areas in femora and vertebrae were smaller in treated rats (p ≤ 0.05), due to the less frequent presence of very large lesions. In untreated animals, osteoclasts appeared to be active at the tumor/bone interface and osseous structures were often completely replaced by expanding tumors. In contrast, metastases in treated animals caused less disruption of skeletal histoarchitecture. The apparent lack of osteoclastic activity and retention of bone within lesions suggested a decreased contribution of osteoclasts to the bone resorptive process. An in vivo immunohistochemical cell proliferation assay failed to reveal differences in the percentage of dividing tumor cells in bone metastatic sites in treated versus untreated animals. The results demonstrate significant effects of risedronate treatment on the incidence and size of observed skeletal metastases in this model.
Comparison of the effects of 3,5,3′-triiodothyroacetic acid and triiodothyronine on bone resorption in cultured fetal rat long bones and neonatal mouse calvariae
Authors: Kawaguchi Hiroshi , Pilbeam Carol C. , Woodiel Florence N. , Raisz Lawrence G.
Published: February 2010

Thyroid hormones can stimulate bone resorption both directly and indirectly by increasing endogenous prostaglandin (PG) synthesis. Because 3,5,3′-triiodothyroacetic acid (Triac, Tiratricol) can bind to nuclear thyroid hormone receptors with high affinity, we compared the effects of Triac and 3,5,3′-triiodothyronine (T3) on bone resorption. 45Ca release was measured at 2, 5, and 8 days from 19-day-old-fetal rat long bones in the presence or absence of calcitonin, indomethacin, aphidicolin (APC), cortisol, or interleukin-1 receptor antagonist (IL-1ra). Both Triac and T3 stimulated 45Ca release in a dose-dependent manner. The maximal treated/control ratio (T/C) for Triac (10−7 M) was 1.6 ± 0.1 at 2 days and 2.5 ± 0.1 at 5 days and, for T3 (10−7 M), 1.5 ± 0.1 at 2 days and 2.6 ± 0.1 at 5 days. These responses were smaller and slower than that to PTH. Significant stimulation of resorption was observed at 10−8, 10−9, and 10−10 M for Triac but only at 10−8 and 10−9 M for T3. Indomethacin (10−6 M) and IL-1ra (1 μg/ml) did not decrease the effects of Triac or T3. In contrast, calcitonin (10−9 M), APC (30 μM), and cortisol could all block the response to these hormones. These results indicated that the resorptive responses were not mediated by endogenous PG or IL-1 but were osteoclast mediated and dependent on DNA synthesis, suggesting that these hormones acted by stimulating replication of osteoclast precursors or other supporting cells. Thyroid hormones were less effective in 7-day-old neonatal mouse calvariae. A small but significant stimulatory effect on 45Ca release was observed with Triac at 10−7 M at 2 and 4 days, but not with T3, and this effect was abrogated by indomethacin. We conclude that Triac stimulates bone resorption with a potency equal to or greater than that of T3. The effects of thyroid hormones were PG-independent in fetal rat long bones and PG-dependent in neonatal mouse calvariae. Since T3 may be converted to Triac in fasting and nonthyroidal illness, we speculate that Triac could mediate bone loss in these catabolic low-T3 states.
Injection of bovine parathyroid poly(A)+ RNA into xenopus oocytes confers sensitivity to high extracellular calcium
Authors: Chen Tsui-Hua , Pratt Stacy A. , Shoback Dolores M.
Published: February 2010

Parathyroid cells detect increments in the extracellular [Ca2+], which lead to substantial increases in intracellular free Ca2+ ([Ca2+]i) and, ultimately, to suppression of parathyroid hormone (PTH) secretion. To determine whether mRNA from parathyroid tissue could confer sensitivity to high extracellular Ca2+, we isolated and injected total bovine parathyroid poly(A)+ RNA into Xenopus laevis oocytes. To assess translational activity of the RNA, PTH released into the media was measured. Intact PTH was detected in the medium for ≤48 h, and injection of increasing amounts of RNA (∼0.5–50 ng/oocyte) led to the release of greater quantities of PTH. We screened for the expression of a putative Ca2+ sensor molecule by measuring 45Ca efflux from preloaded oocytes, in response to raising extracellular [Ca2+] from 0.7 to 5.7 mM. This increment in [Ca2+] stimulated 45Ca efflux by 249 ± 52 cpm over 20 min from eggs injected with parathyroid poly(A)+ RNA (n = 22). This response was significantly greater than 45Ca efflux from any group of controls exposed to the same change in extracellular Ca2+ (p < 0.02), including oocytes injected with either water, cRNA for the platelet-derived growth factor (PDGF) BB receptor, or T cell poly(A)+ RNA. Size-fractionation of poly(A)+ RNA over sucrose gradients demonstrated that mRNA, which induced responsiveness to high extracellular Ca2+, was present in fractions with transcripts of ˜5–9 kB. Injection of these fractions also conferred sensitivity to the presence of Ba2+ or Sr2+ (both at 5 mM) in the media. These findings establish that parathyroid cells express mRNA for a molecule capable of detecting changes in the extracellular divalent cation concentration. This molecule may play a role in secretory responses mediated by Ca2+ in the parathyroid.
Evidence for both generalized and regional low bone mass among elderly women
Authors: Dr. Davis James W. , Ross Philip D. , Wasnich Richard D.
Published: February 2010

The consistency of bone mass measurements across bone sites was examined in a cohort of elderly Japanese-American women. The study included 744 women of mean age 66.6 years (age range 47–82 years) who had bone densitometry measurements at the spine, calcaneus, and distal and proximal radius. The women were classified at the four bone sites as in the lower, middle, or upper bone mass tertile for their age. Slightly more than half (56%) of the women were in the lower tertile at one or more of the bone sites, and such women were usually in the lower category at more than one site. Of the women, 24% were classified in the lower tertile at all four sites. Furthermore, as a group, women classified as in the low bone mass category at any one site had a low average bone mass at all four sites. Prospectively, the number of low bone mass sites predicted the risk of new spine fractures after adjusting for age and the number of spine and nonspine prevalent fractures. The risk increased approximately 1.3-fold for each additional low bone mass site. A subgroup (15%) of the population had marked heterogeneity in bone mass between sites. These women had one or more lower tertile bone mass site(s) and one or more upper tertile bone mass site(s). The results suggest that osteoporosis may occur as either a generalized or as a regional disorder.
Effects of medium acidification by alteration of carbon dioxide or bicarbonate concentrations on the resorptive activity of rat osteoclasts
Authors: Arnett Timothy R. , Boyde Alan , Jones Sheila J. , Taylor M. Louise
Published: February 2010

Little is known about the extracellular conditions or factors that stimulate mature osteoclasts to resorb mineralized tissues. Isolated mammalian osteoclasts are strongly stimulated by protons in HEPES-buffered culture media in the absence of CO2 and HCO3−, but it has been reported that cell-mediated Ca2+ release from bone organ cultures is increased only when media are acidified by reduction of HCO3− concentrations, and not by increasing Pco2 (considered models of metabolic and respiratory acidosis, respectively). We investigated this question using disaggregated rat osteoclasts cultured on dentin slices for 24 h. The number of pits resorbed per osteoclast was stimulated in media acidified by manipulation of either HCO3− or CO2 concentrations. In experiments in which incubator CO2 was varied, resorption was almost abolished in the presence of 2.5% CO2 at pH 7.61 but increased in a stepwise manner up to 1.3 pits per osteoclast when dentin slices were cultured with 10% CO2 at pH 6.97. The depths and widths of pits, measured using a confocal laser reflection microscope, also tended to increase with increasing CO2 and decreasing pH. However, in experiments where pH was lowered by reducing medium HCO3−, pit size decreased, partially offsetting the increased number of pits resorbed per osteoclast. These findings suggest that rat osteoclasts may be more sensitive to stimulation by CO2 acidosis than by HCO3− acidosis, at least in the short term, and may possibly reflect local regulatory processes in bone.
Effects of kistrin on bone resorption in vitro and serum calcium in vivo
Authors: King Kathleen L. , D'Anza James J. , Bodary Sarah , Pitti Robert , Siegel Mark , Lazarus Robert A. , Dennis Mark S. , Hammonds R. Glenn , Kukreja Subhash C.
Published: February 2010

In many cell systems, cell-cell and cell-matrix interactions are mediated by integrins, a family of cell surface heterodimeric glycoprotein receptors. Osteoclast integrins may play a role in the process of bone resorption. Osteoclasts express the αv and β3 subunits of the vitronectin receptor and adhere to a wide range of proteins in vitro, all of which contain the amino acid sequence Arg-Gly-Asp (RGD), an adhesion site recognition sequence common to many protein ligands that bind to integrins. The effect of kistrin, an RGD-containing snake venom protein, on osteoclast-mediated bone resorption was investigated in vivo and in vitro. When kistrin was infused into normocalcemic and hypercalcemic mice, serum calcium was significantly lowered at 3 and 6 h after the start of infusion, indicating an inhibitory effect on osteoclast activity in vivo. In vitro, kistrin potently inhibited bone resorption by isolated rat osteoclasts cultured on slices of bovine bone, and kistrin also inhibited the attachment of 293 cells expressing recombinant human αvβ3 to fibrinogen (IC50 = 1 nM). These results indicate the potential therapeutic use of RGD-containing molecules for hypercalcemia of malignancy or for other disorders associated with bone loss.