bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural and geometric changes in iliac bone: Relationship to normal aging and osteoporosis
Authors: Foldes J. , Parfitt A.M. , Shih M.S. , Rao D.S. , Kleerekoper M.
Published: February 2010

We Measured Indices Of Bone Volume (Cancellous And Cortical) And Bone Surface (Cancellous, Endocortical, And Intracortical) In Intact, Full-Thickness Transiliac Bone Biopsies Obtained From 47 Healthy White Women (23 Premenopausal And 24 Postmenopausal) And 82 Patients With Postmenopausal Osteoporosis. In The Normal Subjects There Was The Expected Loss Of Cancellous Bone With Age, Best Shown By A Reduction In Bone Surface/Tissue Volume, But No Fall In Cortical Thickness With Age Despite A Significant Reduction In Forearm Bone Density Measured By Single-Photon Absorptiometry. Bone Surface/Bone Volume Was About Four Times Higher In Cancellous Than In Cortical Bone, And Cancellous Bone Contributed About One-Third Of The Total Bone Volume And About Two-Thirds Of The Bone Surface When Related To The Core Volume Referent. In The Osteoporotic Patients, Core Width, An Index Of Iliac Bone Thickness At The Biopsy Site, Was Reduced By 10%, But We Could Not Determine Whether This Was The Result Of Compaction Of The Core Or Of Bone Slenderness. All Indices Of Bone Volume, Cortical As Well As Cancellous, Were Significantly Smaller, As Were The Values For Forearm Bone Densitometry; The Relative Deficits At Different Sites Depended On Whether They Were Expressed As Percentages Or As Z Scores. Bone Surface/Bone Volume Was Increased In Both Cancellous And Cortical Bone, But Bone Surface/Tissue Volume Was Reduced In Cancellous Bone And Increased In Cortical Bone. The Proportions Of Total Bone Volume And Surface Contributed By Cancellous And Cortical Bone Were Almost The Same As In Normal Postmenopausal Women. We Reached The Following Conclusions: (1) Age-Related Bone Loss In Normal Subjects Is More Difficult To Detect By Histomorphometry Than By Densitometry. (2) Cortical Bone Loss May Be Slower In The Axial Than In The Appendicular Skeleton. (3) The Amount Of Bone Is Reduced At All Sites In Osteoporotic Patients; The Deficit Is Not Necessarily Greater In Cancellous Than In Cortical Bone But May Be Greater In The Axial Than In The Appendicular Skeleton. (4) Cortical Bone Contributes Relatively Less Volume And Relatively More Surface In The Axial Then In The Appendicular Skeleton. (5) Depending On The Location And The Referent, Bone Surface And Volume May Change In The Same Or In Opposite Directions, With Consequences For The Time Course Of Bone Loss And The Relationships Between Biochemical And Histologic Indices Of Bone Remodeling.
Calcium-deficient diet in ovariectomized dogs limits the effects of 17β-estradiol and nandrolone decanoate on bone
Authors: Dr. Geusens P. , Schot L.P.C. , Nijs J. , Dequeker J.
Published: February 2010

Postmortem measurements by dual-photon absorptiometry of the femur and the second lumbar vertebra in adult dogs indicated bone loss after ovariectomy, which was more pronounced when calcium-deficient diet was given in ovariectomized dogs. This bone loss was nonhomogeneous throughout the femur. Ovariectomy resulted in trabecular and cortical bone loss, and additional calcium-deficient diet resulted in a further highly significant trabecular bone loss at the proximal epiphysis of the femur and in the vertebra. This bone loss was presumably the result of increased bone turnover, as reflected by the highly significant increase in serum alkaline phosphatase. Estrogens could only partially prevent the bone loss induced by calcium deficiency after ovariectomy, and nandrolone decanoate was not effective. We conclude that (1) ovariectomy results in bone loss in adult dogs, (2) this bone loss is more pronounced after calcium-deficient diet, (3) calcium deficiency could be a limiting factor for the preventive effect of estrogens and nandrolone decanoate, and (4) dual-photon absorptiometry allows the evaluation of nonhomogeneous bone loss throughout excised bones.
Risk factors for hip fractures in men: A preliminary study
Authors: Grisso Jeane Ann , Chiu Grace Y. , Maislin Greg , Steinmann William C. , Portale Julia
Published: February 2010

Although hip fracture rates are higher in women than in men, for older men the lifetime risk of fractures of the femur is substantial. Very little is known about risk factors for hip fracture in men. A preliminary case control study was conducted comparing the medical charts of men with first hip fractures with two sets of age-matched controls. The major risk factors for hip fracture that emerged were preadmission ambulatory problems, confusion, heavy alcohol use, and low body mass. Although this study is limited to a medical chart review in a veteran population, these results confirm some of the known associations for hip fracture in women. Further studies in men are recommended.
Bone cell culture in a three-dimensional polymer bead stabilizes the differentiated phenotype and provides evidence that osteoblastic cells synthesize type III collagen and fibronectin
Authors: Majmudar Gopa , Bole David , Goldstein Steven A. , Bonadio Jeffrey
Published: February 2010

We report a novel method to culture chick embryo osteoblasts in vitro. Primary cells were grown from explants of calvaria and then cultured within alginate polymer beads. Enriched cultures of primary osteoblasts were obtained because these cells grow readily within alginate beads but other cell types present in the initial outgrowth from calvarial fragments, such as fibroblasts, do not. A reproducible bone cell phenotype was observed in calvarial cells cultured in the alginate polymer for as long as 8 months. Alginate is a uronic acid monomer that reversibly polymerizes based on the presence or absence of divalent cations. Osteoblasts derived from the alginate beads elaborated and mineralized an extracellular matrix in vitro that contained fibronectin, type III collagen, and type I collagen. The synthesis and deposition of these matrix molecules was also demonstrated in the chick embryo calvaria in vivo. Together, these in vitro and in vivo observations provide the first evidence that type III collagen and fibronectin colocalize with type I collagen during the development of avian membranous bone. They also indicate that the phenotype of chick embryo osteoblasts can be expanded to include the synthesis of fibronectin and type III collagen.
Inhibition of parathyroid hormone secretion correlates with increased incorporation of 32P into phosphatidylinositol and lysophosphatidylinositol
Authors: Bajpai Sarita , Kraft Kelly , Dr. Hamilton James W.
Published: February 2010

We have studied the incorporation of radioactive P (32P) into lipids of bovine parathyroid tissue under conditions of stimulated and inhibited hormone secretion. Utilizing low (0.5 mM) and high (3.0 mM) concentrations of calcium to regulate parathyroid hormone secretion, we initially found that the labeling of the cellular phospholipids with 32P was greater in those tissues incubated in high-calcium medium. Thin-layer chromatography of lipid extracts prepared from tissue incubated in either low- or high-calcium media revealed that the increased incorporation of 32P (high or low) was localized primarily to two phospholipids. To determine whether the increases were due directly to the different calcium concentrations, the experiments were performed in media containing normal calcium concentrations (1.25 mM) and low (0.5) or high (3.0) magnesium concentrations to modulate hormone secretion. The results were identical to those obtained using low and high calcium, indicating that the increased 32P incorporation was not an effect of high calcium but rather correlated with the inhibition of hormone secretion. The use of other secretagogues confirmed this correlation. The identity of the two phospholipids was established, by two-dimensional thin-layer chromatography, to be phosphatidylinositol (PI) and lysophosphatidylinositol (LPI). The correlation of increased 32P incorporation with inhibition of secretion led us next to examine isolated secretory granules from tissues exposed to either high-or low-calcium conditions. Thin-layer chromatography of granule lipid extracts yielded chromatograms containing PI and LPI, and the radioactivity of each was greater in the high-calcium sample than in the low-calcium sample. Our data suggest that a pathway for the synthesis and/or recycling of PI and LPI is present in secretory granules. Taken together, our observations suggest that phosphatidylinositol and lysophosphatidylinositol may play a role, either directly or indirectly, in the secretory pathway.
Relationship between the number of resorbing cells and the amount resorbed in metabolic bone disorders
Authors: Cohen-Solal Martine , Morieux Caroline , Dr. de Vernejoul Marie-Christine
Published: February 2010

The relationship between bone-resorbing cells, assessed by the presence of tartrate-resistant acid phosphatases (TRAP) and morphologic indices of bone resorption, was determined in 29 osteoporotic patients (14 postmenopausal females and 15 males) and 15 dialyzed patients. The number of TRAP-positive cells per unit of cancellous bone area (N.Oc/B.Ar) was higher in dialyzed patients than in those with osteoporosis (16.8 (pminus) 15.3 versus 4.95 (pminus) 2.86, p < 0.05). The amount of bone resorbed at the basic multicellular unit level was estimated by calculating eroded area containing TRAP cells per bone area (E.Ar+/BA). This novel parameter was similar in dialyzed and in osteoporotic patients (41,700 (pminus) 28,400 versus 32,300 (pminus) 24,600). In contrast, trabecular spacing (Tb.Sp) was identical in both metabolic bone diseases. Trabecular width (169 (pminus) 38 versus 127 (pminus) 32 (mu)m, p < 0.05) and bone area were higher in dialyzed than in osteoporotic patients. N.Oc/B.Ar was significantly related to E.Ar+/BA in dialyzed (r = 0.76, p < 0.05) but not in osteoporotic patients. Tb.Sp was significantly correlated to N.Oc/B.Ar and to the number of TRAP-positive cell nuclei per B.Ar (r = 0.44, p < 0.05) in osteoporotic but not in dialyzed patients. This last result shows that in overt osteoporosis with thin trabeculae, trabecular spacing is related to the number of resorbing cells. In contrast, the spacing of thick trabeculae in dialysis osteodystrophy is not dependent on the number of osteoclasts.
Effects of fixation and demineralization on the retention of bone phosphoprotein and other matrix components as evaluated by biochemical analyses and quantitative immunocytochemistry
Authors: Dr. McKee M.D. , Nanci A. , Landis W.J. , Gotoh Y. , Gerstenfeld L.C. , Glimcher M.J.
Published: February 2010

Aqueous tissue processing and demineralization procedures may adversely affect the inorganic mineral phase of a calcified sample and, where mineral and organic constituents interact, may consequently also indirectly alter organic matrix ultrastructure and distribution. In the present work, the effects of demineralization have been investigated on the retention in chicken bone of two phosphoamino acids, O-phosphoserine and O-phosphothreonine, found in bone phosphoproteins proposed to be important in vertebrate mineralization and, more specifically, on the retention and distribution of a 66 kD bone phosphoprotein (66 kD BPP, osteopontin) also implicated in the calcification process. In tibiae fixed initially with 1% glutaraldehyde and then demineralized in 0.5 N HCl, 0.5 N acetic acid, or 0.1 M EDTA (all containing 1% glutaraldehyde), amino acid analyses and quantitative immunocytochemistry revealed that the phosphoamino acid content and the distribution of the 66 kD BPP were essentially the same as in fixed undemineralized controls. However, demineralization slightly altered the ultrastructural appearance of immunolabeled, electron-dense patches of organic material in the bone matrix. In unfixed bone demineralized with any of these acids, there was a substantial loss of phosphoamino acids and the 66 kD BPP from the bone matrix. The relative ability of these acids to extract phosphoproteins from unfixed bone was found to decrease in the order EDTA > HCl > acetic acid. These results emphasize the differential effects on structural components of various demineralization and extraction procedures for biochemical and immunocytochemical studies of biologic tissues. Furthermore, they demonstrate that initial fixation with glutaraldehyde retains phosphoproteins in bone, with or without demineralization, while being adequate for immunocytochemical localization of certain bone matrix proteins and that an understanding of the action of specimen preparation on organic constituents (as well as inorganic components) is essential for accurately describing ultrastructural matrix-mineral relationships.
Parathyroid hormone-related protein inhibits stimulated uterine contraction in vitro
Authors: Dr. Shew Ronald L. , Yee John A. , Kliewer Douglas B. , Keflemariam Yodit J. , McNeill Daniel L.
Published: February 2010

The effect of parathyroid hormone-related protein (PTHrP) fragments 1–34, 38–64, and 67–86 on acetylcho-line-stimulated rat uterine contraction was examined in vitro. In addition, the possibility that PTHrP-(38–64) or (67–86) influenced relaxation caused by PTHrP-(1–34) was also investigated. Contraction of uterine horns was stimulated with 10−6 or 10−5 M acetylcholine. PTHrP-(1–34) reduced the magnitude of acetylcho-line-stimulated uterine contraction. This effect was dose related over a concentration range of 10−9–10−6 M. Neither PTHrP-(38–64) or PTHrP-(67–86) at concentrations of 10−8–10−6 M affected uterine contraction stimulated by 10−6 M acetylcholine. These fragments did not affect the relaxation caused by 10−7 M PTHrP-(1–34). These results demonstrate that (1) PTHrP-(1–34) at 10−6 M influences contraction of the rat myometrium and (2) the muscle relaxant activity of PTHrP is associated with the first 34 N-terminal amino acids.
In vivo induction of bone by recombinant human transforming growth factor β1
Authors: Beck L. Steven , Ammann Arthur J. , Aufdemorte Thomas B. , Deguzman Leo , Xu Yvette , Lee Wyne P. , McFatridge Lorrie A. , Chen Theresa L.
Published: February 2010

A single application of recombinant human transforming growth factor β1 (rhTGF-β1) adjacent to cartilage was found to induce bone formation in rabbit ear full-thickness skin wounds. At doses that optimally promote soft tissue healing, 25–100 ng rhTGF-β1 per wound caused osseous tissue formation starting 21 days after wounding to reach a peak incidence and area of bone formation at day 42. Bone formation was followed by active remodeling, resulting in lower incidence and area of bone formation at days 56 and 70. The early phase of bone formation was located overlying the cartilage and involved perichondrial cells that appeared to differentiate directly into osteoblasts forming bone matrix without a cartilage precursor. Cartilage was replaced with bone at later time points. rhTGF-β1 was able to increase the ratio of osteoblasts to osteoclasts lining the trabecular surface of bone and thus increase the net amount of bone formation. The present studies suggest a potential therapeutic role for rhTGF-β1 in hard tissue repair.
Effects of ipriflavone and its metabolites on a clonal osteoblastic cell line
Authors: Benvenuti Susanna , Tanini Annalisa , Frediani Uliana , Bianchi Sandro , Masi Laura , Casano Rosaria , Bufalino Lidia , Serio Mario , Dr. Brandi Maria Luisa
Published: February 2010

Protective effects of ipriflavone, an isoflavone derivative, in osteoporosis are believed to be caused by the inhibitory action on bone resorption. A direct effect of ipriflavone on bone formation is as yet unknown. Ipriflavone and four of its metabolites (I, II, III, and V) were examined for their effects on parathyroid hormone response, collagen synthesis, alkaline phosphatase activity, and cell proliferation in a clonal cell population of rat osteoblastic cells. Pretreatment of osteoblasts with high concentrations of ipriflavone for 48 h significantly inhibited the cAMP response to parathyroid hormone, producing a shift in the dose-response curve; at lower concentrations metabolites II and III potentiated the cAMP accumulation induced by low doses of parathyroid hormone. The 48 h treatment with metabolite V at the 1 nM dose significantly stimulated collagen synthesis in osteoblastic cells. Ipriflavone and metabolite I showed a biphasic stimulatory action on the alkaline phosphatase activity of osteoblasts, with a maximal effect at the 0.1 and 1 nM doses, respectively. A similar biphasic response was observed with ipriflavone and metabolite I on osteoblastic cell growth, with a maximal effect at the 0.1 nM concentration. These results suggest a direct role of ipriflavone in modulating the synthetic and growth properties of osteoblast-like cells.
Effects of potassium peroxydiphosphate on bone resorption
Authors: Gaffar Abdul , Alander Cynthia B. , Head Raisz Lawrence G.
Published: February 2010

Potassium peroxydiphosphate (KPDP) is a slowly hydrolyzed pyrophosphate analog that can release hydrogen peroxide during hydrolysis. We tested its effects on the resorption of cultured fetal rat long bones as measured by the release of previously incorporated 45Ca, both by direct addition of KPDP to the medium and after preincubation of KPDP with large-molecular-weight resorbing factors followed by dialysis to reduce the KPDP concentration. With direct addition, KPDP at a concentration of 1 mM could inhibit the resortive response to bacterial lipopolysaccharide (LPS), parathyroid hormone (PTH), prostaglandin E2 (PGE2), and mouse recombinant interleukin-1 (mrIL-1). The response to LPS was partially inhibited at 0.3 mM KPDP. Control resorption in the absence of stimulators was also inhibited. Potassium pyrophosphate at 1 mM was less effective as an inhibitor of bone resorption. The inhibitory effects of KPDP did not appear to be due entirely to nonspecific toxicity since partial recovery occurred after it was removed. There was no significant decrease in [3H]thymidine or [3H]proline incorporation into bones incubated with KPDP at 1 mM for 5 days, but [3H]proline incorporation was decreased at 24 h, suggesting that KPDP may have a general inhibitory effect on bone cells. When media with and without stimulators of resorption were incubated overnight at 4°C with KPDP at 5.8 mM and then dialyzed to bring the concentration to below 0.3 mM, the bone-resorbing activity of PTH, LPS, and mrIL-1 was completely lost. This may have been due to the slow release of hydrogen peroxide; however, preincubation with equimolar concentrations of H2O3 caused only partial inactivation of PTH and LPS. Finally, 3% KPDP applied topically in hamsters reduced alveolar bone resorption, indicating that it could interact directly with local pathogenetic factors in vivo. These results indicate that KPDP may be a useful inhibitor of bone resorption. High concentrations of KPDP are required, but it is biodegradable to the normal body constituents, K+ and HPO42-.
Inhibition of bone resorption by a monoclonal antibody that reacts with a 150 kD membrane protein in chicken osteoclasts
Authors: Hentunen Teuvo A. , Lakkakorpi Päivi T. , Rautiala Timo , Väänanen H. Kalervo
Published: February 2010

Bone resorption is a multistep process that includes the maturation of osteoclast precursors, the special attachment of fully differentiated osteoclasts to mineralized bone surface, and the dissolution of inorganic mineral, as well as the breakdown of organic matrix. We have produced a large panel of monoclonal antibodies directed against chicken osteoclasts to obtain specific probes for studying the function of osteoclasts. One of our antibodies, K20, inhibited bone resorption of isolated osteoclasts almost completely. Several pieces of evidence suggested that the antigen detected by this antibody was located in the plasma membrane of the osteoclast. In western blot analysis K20 antibody specifically recognized a 150 kD protein in the medullary bone microsome fraction under reducing and nonreducing conditions. In addition to osteoclasts and some bone and bone marrow mononuclear cells, a positive immunoreaction was seen in the kidney tubules. These data suggest that monoclonal antibody K20 reacts with an osteoclast surface antigen that is functionally important in bone resorption.
Gallium nitrate inhibits bone resorption and collagen synthesis in neonatal mouse calvariae
Authors: Lakatos Peter , Mong Seymour , Stern Paula H.
Published: February 2010

Gallium nitrate (GN) is an agent used in the treatment of hypercalcemia. To more fully characterize the direct actions of GN on bone, we examined its effects on medium calcium, medium β-glucuronidase (β-GLU), and collagen synthesis in control and hormone-stimulated neonatal (4–6 days) mouse calvariae in vitro. GN (10 μg/ml) inhibited parathyroid hormone-stimulated (PTH; 1 nM) calcium release. A 24 h preincubation with 10 μg/ml of GN was required for complete inhibition; partial inhibition was seen with 12 h preincubation; 1, 3, or 6 h was inadequate. A dose-response study showed that with 24 h preincubation, 5, 3, and 1 μg/ml of GN inhibited 81, 62, and 0% of PTH-induced calcium release. The effects of GN on the release of β-GLU generally paralleled those on the release of calcium except that 10 μg/ml of GN stimulated β-GLU release. Collagen synthesis was inhibited 50% by 3 μg/ml of GN, whereas noncollagen protein synthesis was unaffected. With PTH + GN no further decrease was observed. When GN was withdrawn from the medium after 24 h of preincubation, the inhibitory effect on calcium release and β-GLU activity, but not on collagen synthesis, persisted through the 72 h of culture. GN also inhibited the resorption elicited by thyroxine (1 μM) and interleukin-1β (10 nM) but not by 1,25-dihydroxyvitamin D3 (30 pM). Our results indicate that GN is a powerful inhibitor of bone resorption in neonatal mouse calvariae even at low doses. A preexposure time is required to induce the persistent effect, but the continuous presence of GN is not necessary to maintain its effects on bone. Although GN has a primary cellular site of action, the requirement for preculture could suggest that GN could, in addition, make the bone matrix more resistant to resorption.
Salmon stanniocalcin and bovine parathyroid hormone have dissimilar actions on mammalian bone
Authors: Dr. Stern Paula H. , Shankar Geetha , Fargher Robert C. , Copp D. Harold , Milliken Christine E. , Sato Kanji J. , Goltzman David , Herrmann-Erlee M.P.M.
Published: February 2010

Stanniocalcin (STC), a calcium-regulating glycoprotein hormone isolated from the corpuscles of Stannius of salmon, was tested for effects on bone and calcium metabolism in mammalian species (rats and mice). STC generally failed to alter serum calcium of parathyroidectomized rats at concentrations equimolar with effective concentrations of parathyroid hormone (PTH). STC did not increase cAMP in ROS 17/2.8 or UMR-108 osteosarcoma cells, OK kidney cells, fetal rat limb bones, or neonatal mouse calvariae, and similarly failed to increase urinary cAMP in rats. STC did not consistently stimulate resorption in any of the rodent bone culture systems, although variable resorptive responses were elicited in fetal mouse calvariae. The results indicate that this fish hormone has limited, if any, PTH-like activity on calcium metabolism in mammalian systems.
Structural and composition studies on the mineral of newly formed dental enamel: A chemical, X-ray diffraction, and 31p and proton nuclear magnetic resonance study
Authors: Bonar Laurence C. , Shimizu Masaharu , Roberts James E. , Griffin Robert G. , Glimcher Melvin J.
Published: February 2010

The present report describes a study of the development and maturation of the mineral component of dental enamel. We prepared porcine enamel of different stages of maturation, from the very immature enamel of unerupted teeth, with a mineral content of 45%, to fully mature enamel, with a mineral content of ∼99%. We fractionated the less mature enamel by density centrifugation and examined the enamel density fractions and unfractionated enamel by a variety of chemical and physical techniques, including conventional and radial distribution function x-ray diffraction analysis, conventional and Fourier transform infrared spectroscopy, 31P and 1H nuclear magnetic resonance spectroscopy, and chemical analysis. The three most immature preparations, from unerupted teeth, had mineral contents of 45, 67, and 91 and Ca/P molar ratios of 1.41, 1.44, and 1.47. Density distribution histograms of the three fractions show that the early maturation of dental enamel mineral is accompanied by an increase in tissue density, reflecting the increase in mineral content. The density distribution in each sample is relatively narrow, indicating that the maturation process occurs at a fairly homogeneous rate, with all enamel in an anatomically defined zone mineralizing to about the same extent. X-ray diffraction studies indicate that even the least mature, least mineralized of these immature samples is considerably more crystalline than the most mature bone mineral studied and that crystalline perfection of the enamel crystals increases further with maturation. Both the a and c axes of the mineral unit cell expand significantly during early stages of maturation. Solid-state 31P nuclear magnetic resonance spectroscopy studies indicate that dental enamel contains a DCPD-like HPO4 component in an apatitic lattice, similar to the component previously observed in bone and some synthetic calcium phosphates. The proportion of this DCPD-like component decreases with maturation but is readily detectable even in fully mature enamel. The infrared spectroscopic studies indicate that the 3570 cm−1 band ascribed to the OH− group of the hydroxyapatite crystals is absent in the least mature enamel but can be detected and becomes progressively stronger as the enamel becomes more mature. The increase in the content of the OH− groups of the apatite crystals is concomitant with the observed increase in unit cell parameters. Similar studies on very young and very old mature bone of four different species failed to detect the presence of OH− groups.
Preventive effects of clomiphene citrate on estrogen-deficiency osteopenia elicited by LHRH agonist administration in the rat
Authors: Dr. Goulding Ailsa , Fisher Lorryn
Published: February 2010

The estrogen agonist and antagonist clomiphene citrate has been shown to prevent bone loss induced by ovariectomy in the rat. In young women estrogen-deficiency bone loss is a clinical problem associated with the use of luteinizing hormone releasing hormone (LHRH) agonists, such as buserelin, to treat endometriosis. The aim of this study was to determine whether clomiphene citrate (10 mg/kg body weight per week orally) would prevent the osteopenic effect of buserelin (25 μg/kg body weight per day SC) in the rat. Four groups of animals with 45Ca-labeled skeletons were studied for 4 weeks: group A, placebo controls; group B, buserelin; group C, clomiphene; and group D, buserelin + clomiphene. Bone resorption was monitored by measuring the urinary excretion of 45Ca and hydroxyproline. Clomiphene slowed bone breakdown and inhibited the osteopenic effect of buserelin. Total-body calcium values (mean ± SD) were (mg) 2635 ± 181, 2267 ± 85, 2566 ± 126, and 2624 ± 77 in groups A to D, respectively. Osteopenia was present only in group B (P < 0.001). Interestingly buserelin lowered blood 17β-estradiol and uterine weights to a similar extent in the presence and absence of clomiphene. Because clomiphene inhibited estrogen-deficiency bone loss in buserelin-treated rats without depressing the hypoestrogenic action of this LHRH agonist, it is suggested that the use of clomiphene to protect the skeleton during LHRH agonist therapy of endometriosis warrants further study.
X-ray microanalysis of fluoride distribution in microfracture calluses in cancellous iliac bone from osteoporotic patients treated with fluoride and untreated
Authors: Boivin Georges , Grousson Brigitte , Meunier Pierre J.
Published: February 2010

Fluoride is able to augment cancellous bone mass in vertebral osteoporosis but is responsible for osteoarticular side effects in which microfractures are thought to be involved. During healing of these microfractures, a callus is formed all around the cancellous fracture line. Our hypothesis is that in fluoride-treated osteoporotic patients, calluses are bone sites where fluoride is focally deposited at a high concentration, and this could induce a local defect of calcification with a poor healing of microfractures. Our aim was to validate this hypothesis on several calluses following microfractures in undecalcified iliac cancellous bone from six women with osteoporosis (four fluoride treated and two untreated). Histologically normal iliac cancellous bone tissue, taken from a subject having neither fluoride treatment nor microfracture, was also examined. Selected areas, including new woven bone (calluses) and old lamellar bone, were carbon-coated and analyzed using an electron microprobe. Fluoride Kα and calcium Kα radiations were detected with wavelength and energy-dispersive spectrometers, respectively. In old lamellar bone at a distance from microfractures, the fluoride level was similar in normal and untreated osteoporotic patients but was slightly increased in treated osteoporotic patients. In untreated osteoporotic patients, the fluoride level was slightly higher (about 1.2 times) at the site of microfractures (lamellar and woven bone) than in lamellar bone far from such fractures, but fluoride was homogeneously distributed in lamellar and woven bone. In contrast, in treated osteoporotic patients, fluoride content was much higher (about 1.7 times) at the site of microfractures (lamellar and woven bone) than in lamellar bone far from fractures, but fluoride was heterogeneously distributed within the calluses, with the highest level in woven bone and the smallest in adjacent lamellar bone. The present data demonstrate that fluoride is preferentially concentrated in calluses of cancellous microfractures in fluoride-treated osteoporotic patients. It is likely but remains to be proven that this focal increase in fluoride uptake compromises the healing of microfractures.
Modulation of PTH-stimulated osteoclastic resorption by bisphosphonates in fetal mouse bone explants
Authors: Dr. van der Pluijm Gabri , Löwik Clemens W.G.M. , de Groot Henny , Alblas Marcel J. , van der Wee-Pals Lianne J. A. , Bijvoet OLAV L. M. , Papapoulos Socrates E.
Published: February 2010

We examined the effects of the bisphosphonates Cl2MDP, APD, and Me2APD on osteoclastic resorption in the absence and presence of PTH using fetal mouse osteoclast-free bone explants cocultured with fetal liver as a source of osteoclast precursors. Results revealed qualitative and quantitative differencs among the bisphosphonates tested. With Cl2MDP and APD fractional inhibition of resorption (measured as 45Ca release) in the presence of PTH was proportional to that obtained in its absence. In contrast, Me2APD, which is the most potent inhibitor of the three, was found at low concentrations (≤5 × 10−7 M) to enhance the PTH-stimulated osteoclastic resorption. APD as well, at concentrations that could not inhibit resorption, had a similar effect, but Cl2MDP did not. These studies describe a new phenomenon, that low doses of nitrogen-containing bisphosphonates can act synergistically with PTH and enhance osteoclastic resorption. These findings may have clinical implications in the management of patients with increased osteoclastic resorption due to parathyroid overactivity.
Stimulation by parathyroid hormone-related protein and transforming growth factor-α of phosphate transport in osteoblast-like cells
Authors: Pizurki Lara , Rizzoli René , Caverzasio Joseph , Bonjour Jean-Philippe
Published: February 2010

Parathyroid hormone (1–34) [PTH-(1–34)] has been shown to stimulate sodium-dependent phosphate transport (NaPiT) in UMR-106 osteoblast-like cells through a cAMP-dependent mechanism. Whether a synthetic amino-terminal fragment of parathyroid hormone-related protein (PTHrP) or the full-length molecule, which are recognized to interact with the same receptor as PTH, affect NaPiT in the same way is not known. We investigated and compared the effects of bPTH-(1–34), PTHrP-(1–34), and PTHrP-(1–141) on NaPiT and cAMP production in the osteoblastic cell line UMR-106. Each of the three peptides increased cAMP production and exerted a concentration-dependent stimulation of NaPiT after incubation for 4–6 h. We also studied the effect of transforming growth factor-α (TGF-α), which is another tumoral product secreted by certain hypercalcemia-associated tumors, on NaPiT and the TGF-α-induced modulation of the response to PTHrP or PTH. TGF-α caused a 30% stimulation of NaPiT, which remained stable from 6 to 24 h, by a cAMP-independent mechanism. In contrast, TGF-α attenuated cAMP production stimulated by PTH, PTHrP-(1–34), or PTHrP-(1–141). PTHrP or PTH did not further increase NaPiT in TGF-α-treated cells. These results indicate that NaPiT, a possibly important function of osteoblastic cells, was similarly affected by PTH and PTHrP. TGF-α increased NaPiT and modulated in a similar way the effects of both PTH and PTHrP.
1α,25-Dihydroxyvitamin D3 rapidly increases cytosolic calcium in clonal rat osteosarcoma cells lacking the vitamin D Receptor
Authors: Baran Daniel T. , Sorensen Ann Marie , Shalhoub Victoria , Owen Thomas , Oberdorf Annette , Stein Gary , Lian Jane
Published: February 2010

1α,25-Dihydroxyvitamin D3 [1α,25-(OH)2D3] rapidly increases cytosolic calcium in a variety of cell types. Although these rapid effects do not appear to directly involve genome activation, the requirement for the classic vitamin D receptor is unclear. Clonal rat osteosarcoma cells, ROS 17/2.8, respond to 1α,25-(OH)2D3 with an increase in osteocalcin message but ROS 24/1 cells do not. The lack of the receptor for vitamin D in the ROS 24/1 cells has been confirmed by the absence of any detectable vitamin D-receptor complex binding to the vitamin D-responsive element (VDRE) of the osteocalcin gene and the absence of vitamin D receptor mRNA in the cells. Quin-2-loaded ROS 17/2.8 and ROS 24/1 cells were treated with 1α,25-(OH)2D3 in the presence and absence of extracellular calcium and with the inactive epimer, 1β,25-dihydroxyvitamin D3 [1β,25-(OH)2D3]. The 1α,25-(OH)2D3 increased cytosolic calcium in the ROS 17/2.8 and 24/1 cells after 5 minutes in a dose-responsive manner and in the presence and absence of extracellular calcium. Pretreatment of both cell lines with 1β,25-(OH)2D3 for 30 s blocked the hormone-induced rise in cytosolic calcium. The rapid effects of 1α,25-(OH)2D3 on ROS cells with and without the vitamin D receptor and the ability of the inactive epimer to inhibit these effects indicate that the signaling system mediating the hormone's rapid actions is not the classic vitamin D receptor.
Pamidronate Reduces PTH-mediated bone loss in a gene transfer model of hyperparathyroidism in rats
Authors: Mitlak Bruce H. , Rodda Christine P. , von Deck Mercedes D. , Dobrolet Nancy C. , Neer Robert M. , Dr. Nussbaum Samuel R.
Published: February 2010

We Evaluated Spinal And Femoral Bone Mass And Density Utilizing Dual-Energy X-Ray Absorptiometry (Dexa) In Rats In Which Severe Hyperparathyroidism Was Produced By The Expression Of The Gene For Human Pth-(1–84) (Hpth). This Gene Was Incorporated Into A Retroviral Vector That Was Transfected Into Fibroblasts Which Were Subsequently Injected Into Their Peritoneal Cavities. Further, We Examined The Effect Of The Administration Of Pamidronate On Bone Mass And Density In The Presence Of Extremely High Concentrations Of Hpth. Three Groups Of Rats Were Studied. Groups 1 And 2 Receive The Hpth-Secreting Fibroblasts; Group 2 Subsequently Received Pamidronate (2.5 Mg/Kg Iv) 18 And 27 Days After Receiving The Fibroblasts. These Animals Developed Levels Of Hpth > 1.0 μg/Liter And Became Hypercalcemia Within 20 Days. These Animals Became Lethargic And Were Significantly Lower In Weight Than Age-Matched Controls (Group 3, P < 0.05). After Accounting For The Animal Weight There Was A Further Significant Decrease In Bone Mineral Content And Density (Bmc And Bmd) On Day 29 Attributable To Hpth-Mediated Bone Loss. Treatment With Pamidronate Resulted In A Higher Bmc Of The Lumbar Spine Than In The Untreated Animals, With Elevated Concentrations Of Hpth. The Bmd Was Significantly Higher At Both The Lumbar Spine And Femur In The Pamidronate-Treated Animals (P < 0.05). The Cv Of Paired Measurements Of Bmd Was 2.7% At The Spine And 1.5% Of A Femur, Respectively. The Bmc Of The Lumbar Spine And Femur Was Closely Correlated With The Ashed Weight Of The Same Bones (R = 0.92 And 0.85, Respectively).
A new method for measuring cancellous bone erosion depth: Application to the cellular mechanisms of bone loss in postmenopausal osteoporosis
Authors: Cohen-Solal Martine E. , Shih Mei-Shu , Lundy Mark W. , Dr. Parfitt Michael A.
Published: February 2010

We have devised a new method for measurement of final depth of erosion in cancellous bone with an intra-individual precision of 4.3% and applied it to determine the mechanism of continuing reduction in trabecular thickness after menopause. Mean erosion depth (SD) was 40.8 (2.0) μm in 10 healthy postmenopausal women and 41.4 (2.1) μm in 10 age-matched patients with postmenopausal osteoporosis; the difference was not statistically significant. In contrast, wall thickness, using a method based on density differences between new and old bone, was 39.5 (2.0) μm in the normal subjects and 35.3 (2.0) μm in the patients with osteoporosis (p < 0.0001). The balance per remodeling cycle (ΔBMU) was −1.34 (2.49) μm in the normal subjects and −6.11 (1.95) μm in the patients with osteoporosis. This difference was also highly significant (p < 0.001). Indirect estimations of erosion depth and ΔBMU, based on the fall in trabecular thickness from an assumed premenopausal value of 147 μm and the number of remodeling cycles accumulated since menopause, agreed closely with the measured values. Erosion depth measured by the Eriksen method also showed no significant difference between the two groups, but because the values were substantially higher ΔBMU was improbably high in both groups, did not differ significantly between groups, and was inconsistent with the observed difference in trabecular thickness. We conclude that (1) the more rapid continuing loss of cancellous bone in patients with postmenopausal osteoporosis than in age-matched control subjects is due entirely to a difference in wall thickness, not to a difference in erosion depth; and (2) defective recruitment and/or function of osteoblasts is the major cellular mechanism of trabecular thinning in patients with postmenopausal osteoporosis and probably also in normal subjects. We emphasize that these conclusions do not speak to the mechanism of complete removal of trabeculae in the early years after menopause.
Loading-related increases in prostaglandin production in cores of adult canine cancellous bone in vitro: A role for prostacyclin in adaptive bone remodeling?
Authors: Rawlinson Simon C.F. , El-Haj Alicia J. , Minter Sarah L. , Tavares Ignatius A. , Bennett Alan , Dr. Lanyon Lance E.
Published: February 2010

Cyclic mechanical loading sufficient to engender strains of physiologic magnitude applied to recently excised canine cancellous bone cores in vitro increased the release of prostaglandin E (PGE) and prostacyclin (PGI2, measured as its breakdown product 6-keto-PGF1α), during a 15 minute loading period in which PG levels were measured in perfusing medium at 5 minute intervals. Peak production occurred in the 0–5 minute sample. Mean levels preload compared to during load were PGE, 2.66 and 3.67 ng/ml (p < 0.002); and 6-keto-PGF1α, 543 and 868 pg/ml (p < 0.007). The elevated levels then declined to preload levels during the loading period. However, the 5–10 minute but not the 10–15 minute samples still contained levels greater than preload values. A second 15 minute period of load, 1 h following the end of the first, produced smaller increases in the levels of release that were statistically significant only for the first 0–5 minute sample during load (preload compared to load mean values, PGE, 1.09–1.66 ng/ml, p < 0.02; 6-keto-PGF1α, 401–558 pg/ml, p < 0.04). Immunolocalization revealed PGE and 6-keto-PGF1α in lining cells and 6-keto-PGF1α but not PGE in osteocytes. Addition to the medium of 1 μM PGE2, approximating the concentration produced by loading, had no significant effect on the specific activity of the extractable RNA fraction labeled with [3H]uridine, whereas 1 μM PGI2 produced an increase similar to that seen previously with loading. These results suggest that both PGE and PGI2 may be involved in loading-related adaptive bone modeling and remodeling. The presence of the PGI2 breakdown product in osteocytes and the RNA response to PGI2 suggest that this prostanoid may be an early factor in the cascade of events between strain in the bone matrix and subsequent osteoregulatory responses performed by bone cell populations.
Bone morphogenetic proteins (BMP-2 and BMP-3) promote growth and expression of the differentiated phenotype of rabbit chondrocytes and osteoblastic MC3T3-E1 cells in vitro
Authors: Hiraki Yuji , Inoue Hiroyuki , Shigeno Chohei , Sanma Yuji , Bentz Hanne , Rosen David M. , Asada Akira , Suzuki Fujio
Published: February 2010

We studied the effects of highly purified bone morphogenetic protein 2 and 3 (BMP-2 and −3) on growth plate chondrocytes and osteoblastic cells in vitro and compared to TGF-β. A mixture of BMP-2 and 3 (BMPs) strongly stimulated DNA synthesis of chondrocytes in the presence of fibroblast growth factor (FGF). BMPs induced rapid maturation of chondrocytes at a growing stage: BMPs transformed the cells into rounded cells and induced marked accumulation of cartilage matrix; TGF-β slightly reduced matrix accumulation and changed cell morphology into spindle-like in the presence of FGF. Moreover, exposure of chondrocytes to BMPs resulted in a dramatic increase of the putative ˜80 kD PTH receptors expressed on the cell surface. In multilayered chondrocytes at the calcifying stage, BMPs stimulated alkaline phosphatase (ALPase) activity but TGF-β inhibited it. In osteoblastic MC3T3-E1 cells, BMPs were found to be the most potent stimulator of ALPase activity thus far described: ALPase in the cells treated with ˜100 ng/ml of BMPs reached 5- to 20-fold over the basal, whereas TGF-β inhibited expression of ALPase activity in these cells. The stimulatory action of BMPs overrode the inhibition of ALPase activity by TGF-β when the cells were incubated with TGF-β and BMPs. BMPs also upregulated expression of the ˜80 kD PTH receptor on the cells. These results suggest that BMPs have unique biologic activities in vitro that lead to growth and phenotypic expression of cells playing a critical role in endochondral bone formation.
Journal of Bone and Mineral Research
Authors: Marx Stephen J.
Published: February 2010

Primary hyperparathyroidism is caused by defects in the parathyroid gland. Investigations have implicated three interesting genes whose mutation can cause primary hyperparathyroidism. Familial hypocalciuric hypercalcemia is believed to be an atypical form of primary hyperparathyroidism with an inherited defect in calcium recognition expressed not only in all parathyroid chief cells (thus a polyclonal defect) but in some renal tubular cells as well. In typical primary hyperparathyroidism a monoclonal parathyroid tumor is usually the central cause. Either of two apparently different genes on the long arm of chromosome 11 has been implicated in development of a parathyroid tumor clone. One gene (D11S287) was shown to have undergone a rearrangement with the parathyroid hormone gene on the short arm of the same chromosome (pericentro-meric inversion) in a small fraction of tumors; the D11S287 locus may encode a growth stimulator. Another gene, the locus for familial multiple endocrine neoplasia type 1 (FMEN1), is likely to encode a growth inhibitor. Inactivation of this gene or another nearby gene by somatic mutation has been indirectly implicated in one-quarter of sporadic parathyroid adenomas and in more than half of parathyroid tumors in FMEN1. In conclusion, studies have suggested three different mechanisms for parathyroid gland dysfunction in primary hyperparathyroidism: (1) a defect in calcium recognition, (2) a monoclonal tumor from overexpression of a growth stimulator, or (3) a monoclonal tumor from inactivation of a growth inhibitor.
Journal of Bone and Mineral Research
Authors: Lafferty Frederic W.
Published: February 2010

The differential diagnosis of hypercalcemia has expanded to over 25 separate disease states, with primary hyperparathyroidism and malignancy accounting for 80–90% of all hypercalcemic patients. Primary hyperparathyroidism comprises the majority of hypercalcemic patients among the ambulatory population, but malignancy accounts for up to 65% of such patients in the hospital. Factors favoring primary hyperparathyroidism include a family history of hyperparathyroidism or multiple endocrine neoplasia, a history of childhood radiation to the head and neck, the postmenopausal state, a history of renal calculi or peptic ulcer, hypertension, the induction of hypercalcemia by thiazides, or an asymptomatic patient with a prolonged, stable mild hypercalcemia. The usefulness of the serum calcium, parathyroid hormone, chloride, phosphorus, serum 25-OHD, and l,25-(OH)2D, and urinary calcium in the differential diagnosis of hypercalcemia is discussed. The pitfalls of an excessive reliance on the serum PTH in diagnosing hyperparathyroidism are stressed. The discriminant values of the serum calcium, chloride, phosphorus, and parathyroid hormone are explored, with the serum parathyroid hormone, chloride, and calcium proving most useful in separating primary hyperparathyroidism from other forms of hypercalcemia. Multivariate discriminant analysis using the serum calcium, phosphorus, and chloride and the hematocrit achieves an accuracy of 95–98% and is the most economical method of identifying hyperparathyroidism. The addition of the amino-terminal or intact PTH assay increases the accuracy to 99% and is essential in the presence of renal insufficiency.
Journal of Bone and Mineral Research
Authors: Ott Susan M.
Published: February 2010

Bone disease is one of the most important clinical manifestations of primary hyperparathyroidism. Recent studies have shown that the loss of bone in “modern” hyperparathyroidism is not homogeneous throughout the skeleton. Whereas measurements of the spine and the trabecular portion of iliac crest are normal or even slightly increased, the wrist and the cortical bone of the iliac crest are significantly lower than expected. Thus, knowledge of the percentage of trabecular bone at each measurement site is important. These include distal radius, 80%; midradius, 5%; vertebrae, 19–25%; vertebral body, 33–42%; and femoral neck, 43%. Knowledge of the precision and its relationship to ranges and rates of change of bone mass are essential. For an individual, the bone mass should change by 2.8 times the precision before one can determine with 90% confidence that the change was real, not due to measurement error. Noninvasive methods of measuring bone mass that are commercially available include single- and dual-photon absorptiometry, quantitative computed tomography, and dual-energy x-ray absorptiometry. The precision of these techniques varies, with the best values reported using dual-energy x-ray absorptiometry. Research should be done to describe more completely the patterns of bone loss in this disease, including longitudinal studies on rates of loss at different skeletal sites. Studies that relate the bone mass at a particular site to fractures must be done in populations with hyperparathyroidism to see if the risk of fracture is similar to the risk in normal or osteoporotic individuals.