bluuebunny/crossref_metadata_2025 huggingface dataset:
The influence of Vitamin D metabolites on the calcification of cartilage matrix and the C-propeptide of type II collagen (Chondrocalcin)
Authors: Hinek Aleksander , Dr. Poole A. Robin
Published: February 2010

The influence of vitamin D metabolites (at 1 × 10−10M) on the calcification of cartilage matrix (measured by 45Ca2+ uptake) and the C-propeptide of type II collagen (measured by radioimmunoassay) has been studied using organ cultures and chondrocytes isolated from growth plates of vitamin D-deficient and -sufficient 11-day-old rats. Vitamin D-deficient rats had reduced amounts of C-propeptide in their serum and freshly isolated growth plate chondrocytes. In all chondrocytes cultured from vitamin D-deficient animals, the C-propeptide content was maximal at 24 hr whereas calcification continued to increase for up to 72 hr. In organ and chondrocyte cultures of tissue from vitamin D-sufficient rats, both 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25(OH)2D3) were required for maximal stimulation of calcification and maximal increases in C-propeptide content. In these D-replete tissues, 24,25-(OH)2D3 had a less stimulatory effect on both calcification (organ and cell cultures) and C-propeptide (organ cultures only), while 1,25(OH)2D3 alone had no effect in cell cultures but an inhibitory effect in organ cultures.

In all of these studies, maximal stimulation by vitamin D metabolites of 45Ca2+ incorporation was always accompanied by a maximal net increase in CPII content. Since increases were often quantitatively and temporally different, if would appear that the C-propeptide does not simply accumulate by a process of passive binding to mineral but that its increased concentration is the result of an active process that may be causally related to calcification.

These observations clearly demonstrate that 24,25(OH)2D3 is alone required for maximal calcification of cartilage matrix in growth plate cartilages of vitamin D-deficient rats and that this metabolite also produces maximal increases in the synthesis of the C-propeptide. Moreover, 1,25(OH)2D3 is required with 24,25(OH)2D3 for the maximal calcification and maximal increases in the amount of C-propeptide which are observed in vitamin D-sufficient animals.
Characterization of specific thyroid hormone receptors in bone
Authors: Dr. Krieger Nancy S. , Stappenbeck Thad S. , Stern Paula H.
Published: February 2010

Thyroid hormones stimulate bone turnover in vivo and increase Ca release from bone in vitro. To investigate further the effects of thyroid hormones in bone, we have characterized specific nuclear receptors for [125]triiodothyronine (T3) in neonatal mouse calvaria. Maximal specific binding of [125I]T3 to isolated nuclei occurred within 60 min at 22°C. [125I]T3 binding was completely and rapidly displaced by the addition of 10−6M unlabeled T3; the dissociation appears to be first order with t1/2 = 36 min. The IC50 for competition by unlabeled T3 was approximately 10−8M. The relative affinity of thyroxine (T4) for the receptor was ˜10 × lower than T3, consistent with its lower biological potency in most target tissues for thyroid hormones. Only weak competition was observed with diiodotyrosine at concentrations up to 10−4M. We have previously shown that the cardiotonic agent milrinone stimulates bone resorption in vitro with characteristics similar to those of T4. Structural homology between milrinone and T4 was recently reported. Milrinone, like diiodotyrosine, was only a weak competitor for binding at concentrations up to 10−4M. Milrinone inhibited collagen synthesis in the calvaria. The results suggest that the effects of milrinone on bone turnover in calvaria in vitro are probably not mediated through a thyroid hormone receptor.
Fluoride content in human iliac bone: Results in controls, patients with fluorosis, and osteoporotic patients treated with fluoride
Authors: Boivin Georges , Chapuy Marie-Claire , Baud Charles A. , Meunier Pierre J.
Published: February 2010

The major part of fluoride ingested is fixed on calcified tissues, mainly in bone tissue, and then is progressively but slowly recycled during bone remodeling. Thus, the measurement of bone fluoride content allows the determination of the extent of bone fluoride retention, and this parameter constitutes a useful complement to bone histology for the diagnosis of skeletal fluorosis and could also be used for the management of fluoride treatment of osteoporosis. A simple method is described to measure the fluoride content in calcined human iliac bone samples. Bone ashes were diluted in perchloric acid, and the measurement of the bone fluoride content was performed using a specific ion electrode combined with a reference electrode. Reference values are given for bone tissue from 76 control subjects (0.08 ± 0.05% of bone ash), from two groups of 117 and 102 untreated osteoporotic patients (0.05 ± 0.03% and 0.08 ± 0.05%, respectively), from 166 sodium fluoride-treated osteoporotic patients (mean bone fluoride content varying from 0.24 to 0.67%, depending on the duration of therapy), and from 96 patients showing typical skeletal fluorosis (mean bone fluoride content varying from 0.56 to 1.33%, depending on the etiology of fluorosis and the relationship with the amount of fluoride ingested as well as with the duration of fluoride exposure). During a prolonged exposure of adult bone tissue to fluoride, the early bone fluoride uptake is variable and depends on the remodeling activity; then it increases rapidly before becoming more or less stable at a maximum level.
Early effects of parathyroid hormone on membrane potential of rat osteoblasts in culture: Role of cAMP and Ca2+
Authors: Fritsch Jeanine , Edelman Aleksander , Balsan Sonia
Published: February 2010

Microelectrodes were used to investigate the possible involvement of cAMP and Ca2+ ions in the parathyroid hormone's, bPTH(1–34), effect on the membrane potential of rat osteoblasts in primary culture. Parathyroid hormone (10−7 M) depolarized cell membrane by 25.0 ± 6.1 mV (mean ± standard deviation, SD; n = 17). Blocking Ca2+ influx with the Ca channel blocker cobalt revealed two phases in the hormone effect: a rapid and slight membrane hyperpolarization followed by sustained depolarization. In addition, cobalt significantly (p < 0.01) decreased the magnitude of the PTH depolarizing action. The addition of dibutyryl-cAMP (10−3 M) to the perfusion solution also resulted in a biphasic effect. At a lower concentration (10−4 M), dibutyryl-cAMP produced only membrane hyperpolarization, suggesting a cAMP dose dependence of the opposite membrane potential changes. Forskolin (10−5 M) and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) (10−4 M) mimicked the depolarizing effect of PTH. IBMX at a low concentration (5 × 10−6 M) potentiated the depolarizing effect of PTH. Increases in [Ca2+]i using Ca2+ ionophore A23187 and intracellular injection of CaCl2 or inositol trisphosphate decreased the PTH depolarizing action, whereas intracellular injection of EGTA enhanced this effect. These results indicate that PTH evokes a biphasic change in rat osteoblast membrane potential that seems to be mediated by an increase in cAMP and modulated by intracellular calcium.
Cellular basis of inflammation-induced osteopenia in growing rats
Authors: Krempien B. , Vukičević S. , Vogel M. , Stavljenić A. , Böchele R.
Published: February 2010

Local nonosseous inflammation provoked by four subcutaneous talc powder injections induced a marked trabecular bone loss in rats within 7 days. The disturbance included suppression of bone elongation, inefficiency and decreased number of trabecular osteoblasts, decreased osteoprogenitor cell number in tibial metaphyses, and bone marrow expansion.

Neither the appearance and function of osteoblasts in the vicinity of the cortical bone nor the number of osteoclasts in the metaphysis were found to be altered. The loss of trabecular bone in granulomatosis was based on a suppression of bone elongation and a failure of osteoblasts to form normal secondary spongiosa.
Nucleotide sequence of cloned cDNAs encoding chicken preproparathyroid hormone
Authors: Khosla Sundeep , Demay Marie , Pines Mark , Hurwitz Shmuel , Potts John T. , Kronenberg Henry M.
Published: February 2010

In order to characterize an avian parathyroid hormone gene, a λgt10 cDNA library constructed from chicken parathyroid gland mRNA was screened with a human preproparathyroid hormone (preproPTH) cDNA probe. Nucleotide sequence analysis of three independent clones confirmed that they encoded chicken preproPTH. This analysis, complemented by primer extension and Northern blot analysis of mRNA, demonstrated a 5′-untranslated region for chicken preproPTH of 127 nucleotides, a coding region of 357 nucleotides, and a 3′-untranslated region of approximately 2500 nucleotides. The coding sequence predicts a mature chicken PTH of 88 amino acids in contrast to the 84 amino acids of the mammalian hormones. Comparison of the avian and the mammalian hormones shows striking homology in the region of amino acids 1–32. The middle and carboxyl-terminal portions of chicken PTH, however, differ considerably from the mammalian hormones and include deletions of sequences conserved in mammalian PTH and insertions of novel peptide sequences. Comparison of the avian and mammalian structures suggests potential alterations of the mammalian sequences that may lead to altered bioactivity and/or hormone metabolism.
Effects of infusion of human parathyroid hormone-related protein-(1–40) in nude mice: Histomorphometric and biochemical investigations
Authors: Rosol Thomas J. , Capen Charles C. , Horst Ronald L.
Published: February 2010

Female nude mice were infused with 5.0, 3.0, or 1.0 μg/day of synthetic human parathyroid hormone-related protein (PTHrP) or control diluent with subcutaneous Alzet miniosmotic pumps for 7 days. Serum calcium was increased (p < 0.01) on days 3 (13.9 mg/dl), 5 (13.6 mg/dl), and 7 (12.9 mg/dl) in mice infused with PTHrP at 5.0 μg/day compared with control nude mice (8.8 mg/dl). Serum calcium was significantly increased to a lesser degree in mice infused with 1.0 μg/day PTHrP (day 3) or 3.0 μg/day (days 3 and 7). Serum phosphorus was decreased (p < 0.01) in all three groups of mice infused with PTHrP (4.6 mg/dl, 5.0 μg/day; 6.7 mg/dl, 3.0 μg/day; and 6.4 mg/dl, 1.0 μg/day) compared with controls (8.5 mg/dl). Serum 1,25-dihydroxycholecalciferol was increased (2.4-fold) in mice infused with PTHrP (5.0 and 3.0 μg/day). The urinary calcium-creatinine ratio (0.74 compared with 0.034 in controls) was increased (p < 0.03) in mice infused with PTHrP (5.0 μg/day), but the urinary phosphorus-creatinine ratio was not different from that in controls. The urinary cAMP-creatinine ratio was increased (1.6-fold) in mice infused with PTHrP (5.0 μg/day). Static bone histomorphometry revealed increased (p < 0.01) trabecular bone area, osteoblast perimeter, osteoid perimeter, osteoid width, wall width, osteoclast area, number of osteoclasts, and osteoclast perimeter in trabecular bone of lumbar vertebrae from mice infused with PTHrP. Dynamic bone histomorphometry demonstrated increased (p < 0.01) double-labeled perimeter, mineralizing perimeter, and bone formation rate. The results of this study indicated that PTHrP increased serum calcium and 1,25-dihydroxy-cholecalciferol, decreased serum phosphorus, increased urinary excretion of calcium, phosphorus, and cAMP, and increased both bone resorption and formation in nude mice. PTHrP mimics the action of native PTH in vivo and is likely to be an important protein in the pathogenesis of humoral hypercalcemia of malignancy.
Parathyroid hormone regulation of proline uptake by cultured neonatal mouse osteoblastlike cells
Authors: Yee John A. , Sutton Julie K.
Published: February 2010

Regulation of proline uptake by the synthetic amino-terminal fragment of bovine parathyroid hormone [bPTH-(1–34)] has been studied in confluent primary cultures of osteoblastlike cells isolated from neonatal mouse calvaria. The initial velocity of proline transport was increased by 85% in cultures treated with 24 nM bPTH-(1–34) for 6 h. Cycloheximide, at a concentration that inhibited protein synthesis by 97%, did not prevent this effect. However, adding the inhibitor during the first 1–2 h of hormone treatment did significantly reduce its magnitude. Exposure of cells to the inhibitor alone caused a time-dependent decrease in the basal rate of proline uptake. In the absence of protein synthesis, the maximal velocity (Vmax) of transport was 60% greater in cultures treated with 24 nM bPTH-(1–34) than in controls. The concentration of proline at which half-maximal transport occurred (Km) was unchanged. In cultures treated with cycloheximide alone, proline transport decreased as a first-order exponential with a half-life of 250–280 min. Parathyroid hormone significantly reduced this decline, increasing the half-life of proline transport activity about fourfold. These effects were duplicated by 1 mM DBcAMP. It is concluded that bPTH-(1–34) increases proline transport in osteoblastlike cells by decreasing the degradation of amino acid transport system A proteins. The hormone may also affect the synthesis of these molecules. These effects appear to be mediated by cAMP.
Contrasting mechanisms of hypercalcemia in patients with early and advanced humoral hypercalcemia of malignancy
Authors: Dr. Ralston Stuart H. , Boyce Brendan F. , Cowan Robert A. , Gardner Mary D. , Fraser William D. , Boyle Iain T.
Published: February 2010

The mechanisms of hypercalcemia were assessed in 15 patients with humoral hypercalcemia of malignancy (HHM) who had tumors at various stages of progression. In patients with early tumors, bone biopsies were generally normal and the hypercalcemia was due to an elevation in renal tubular resorption of calcium. Conversely, osteoclastic resorption was markedly increased in patients with advanced tumors, particularly those in whom the biopsies were obtained postmortem. Osteoclast surface (Oc.S) correlated positively with the stage of tumor progression (r = 0.80, p < 0.002), degree of immobility (r = 0.87, p < 0.002), and level of urinary cyclic AMP excretion (r = 0.60, p < 0.02). When compared with a group of ambulant patients with primary hyperparathyroidism (HPT), osteoblast surface (Ob.S%) in HHM was depressed (median and range): 1.2% (0–11.6%) versus 5.3% (1.1–32.0%) (p < 0.001). However, a relatively low Ob.S (4%) and raised Oc.S (43.5%) were also seen in an immobilized patient with severe HPT.

These data suggest that the PTH-related peptides currently invoked in the pathogenesis of HHM may initially cause hypercalcemia by enhancing renal tubular calcium resorption. The increase in osteoclastic activity and depression of osteoblastic activity that subsequently occurs is probably due to the combined effects of immobilization and higher circulating levels of PTHrP on the skeleton. However, the release of other bone-resorbing factors by the tumor, which have a depressant effect on osteoblastic activity, remains possible.
A direct effect of 24,25-(OH)2D3 and 1,25-(OH)2D3 on the modeling of fetal mice long bones in vitro
Authors: Schwartz Z. , Soskolne W. A. , Atkin I. , Goldstein M. , Ornoy A.
Published: February 2010

To examine the effects of 24,25-(OH)2D3 and 1,25-(OH)2D3 on fetal long bone modeling the radii and ulnae of 16 day fetal mice were grown in vitro for 2 days. Their growth, mineralization, and resorption were assessed by measuring diaphyseal length, calcium and phosphorus content, hydroxyproline-protein ratios, and the release of incorporated 45Ca. The results showed that 24,25-(OH)2D3 at concentrations of 10−10-10−8 M stimulated the growth of the bones as indicated by their increased diaphyseal length, periosteal bone area, and hydroxyproline content. Calcium and phosphorus content was significantly increased; 45Ca release was unaltered. Bones incubated in media containing 10−6 M 24,25-(OH)2D3 responded in a similar fashion to bones incubated in media containing 10−10-10−8 M 1,25-(OH)2D3, with inhibition of bone growth as indicated by reduced diaphyseal length, periosteal bone area, hydroxyproline-protein ratios, and calcium and phosphorus content; 45Ca release was significantly increased. Neither metabolite affected total bone length. The results suggest a role for 24,25-(OH)2D3 in the growth of fetal mice bones in vitro and also confirm the findings from previous studies that 1,25-(OH)2D3 and high concentrations of 24,25-(OH)2D3 stimulate bone resorption.
Bone matrix stimulates osteoclastic differentiation in cultures of rabbit bone marrow cells
Authors: Fuller Karen , Chambers Timothy J.
Published: February 2010

Cells showing osteoclastic characteristics have not been identified outside bone. Because osteoclasts originate from an extraosseous source, this suggests that identifiable osteoclastic features do not develop until the precursors enter bone, where the local microenvironment may signal osteoclastic differentiation or maturation. We assessed the influence of bone matrix on osteoclastic differentiation by incubating bone marrow cells, after removal of pre-existing osteoclasts, on plastic coverslips or slices of devitalized cortical bone. We found that there was a threefold increase in the number of osteoclast-specific MAb-positive cells on the bone matrix compared with plastic coverslips. The number of MAb-positive cells correlated with the extent of excavation of the surface of the bone slices. Multinuclearity correlated with MAb-positive cell density, and for any given density the proportion of MAb-positive cells that were multinucleate was similar on plastic and bone. We conclude that, in the presence of 1,25-(OH)2 vitamin D3, bone matrix stimulates the generation of osteoclasts but has no demonstrable influence on the fusion of mononuclear osteoclastic precursors.
Effects of vitamin D metabolites on collagen production and cell proliferation of growth zone and resting zone cartilage cells in vitro
Authors: Schwartz Z. , Schlader D. L. , Ramirez V. , Kennedy M. B. , Dr. Boyan B. D.
Published: February 2010

Previous studies have suggested that vitamin D metabolites directly influence the differentiation and maturation of chondrocytes in calcifying cartilage. Recently, this laboratory has shown that the response of chondrocyte plasma membrane and matrix vesicle enzymes to 1,25-(OH)2D3 and 24,25-(OH)2D3 is both cell and membrane specific. The current study demonstrates that cell replication and matrix protein synthesis are also modulated by vitamin D. Confluent, third-passage growth zone (GC) and resting zone (RC) costochondral chondrocytes were incubated in medium containing 10−13-10−7 M 1,25-(OH)2D3 or 10−12-10−6 M 24,25-(OH)2D3. The amount of collagenase-digestible protein (CDP) secreted into the media was inversely proportional to the concentration of fetal bovine serum (FBS). At 10% FBS, greater than 80% of the CDP was incorporated into the matrix. 1,25-(OH)2D3 stimulated CDP and percentage collagen synthesis by GC cells but had no effect on the synthesis of noncollagenous protein (NCP). 1,25-(OH)2D3 inhibited CDP and percentage collagen synthesis by RC cells but did not alter NCP synthesis. [3H]thymidine incorporation was inhibited in both cell types, whether confluent or subconfluent cultures were examined. At 10−6 and 10−7 M 24,25-(OH)2D3, there was a significant decrease in CDP production and percentage collagen synthesis by RC cells but no effect on NCP. However, at 10−9 and 10−10 M hormone there was an increase in NCP production but no effect on CDP, resulting in a decrease in percentage collagen synthesis. CDP and NCP production were unaffected by 24,25-(OH)2D3 in GC cells. High concentrations of hormone inhibited [3H]thymidine incorporation in both cell types. 24,25-(OH)2D3 also stimulated [3H]uridine incorporation at 10−8 and 10−9 M in RC cells. These data support the hypothesis that vitamin D metabolites influence the development and maturation of calcifying cartilage and that the response of cells to hormone is dependent on their state of differentiation.
Effect of arachidonic acid metabolites on bone resorption by isolated rat osteoclasts
Authors: Fuller Karen , Chambers Timothy J.
Published: February 2010

Arachidonic acid metabolites (eicosanoids) have major effects on bone but their role is unclear. Many are known to stimulate bone resorption in organ culture, but paradoxically, previous work has suggested that at least some of them act as direct inhibitors of osteoclastic function. In an attempt to clarify the role of eicosanoids in bone physiology, we have defined the duration of action and relative potencies of prostaglandin (PG) E1and E2 and have extended the range of eicosanoids tested on isolated osteoclasts. We have found that PGE1 and PGE2 inhibited bone resorption by isolated osteoclasts for at least 6 h. Inhibition was followed by recovery to control, not supranormal levels. Bone resorption was inhibited in the range 10−5-10−9 M for PGE1 and PGE2, and the rank order as resorption inhibitors was PGE1 > 6-keto PGE1 > PGE2 > PGA2 > PGB2. None of the products of lipoxygenase metabolism showed a significant direct effect. The effects of PGE1 and PGE2 were not antagonistic. Prostaglandin production does not seem to be implicated as a second messenger for the action of calcitonin. Although inhibition of osteoclasts by PGs was less prolonged than that observed in the presence of calcitonin, the sensitivity of osteoclasts to inhibition by PGs, and the duration of the effect without subsequent direct stimulation, suggests that inhibition of osteoclastic resorption is a major physiological role of PG production in bone.
Stimulation of experimental endochondral ossification by low-energy pulsing electromagnetic fields
Authors: Aaron Roy K. , Ciombor Deborah McK. , Jolly Grant
Published: February 2010

Pulsed electromagnetic fields (PEMFs) of certain configuration have been shown to be effective clinically in promoting the healing of fracture nonunions and are believed to enhance calcification of extracellular matrix. In vitro studies have suggested that PEMFs may also have the effect of modifying the extracellular matrix by promoting the synthesis of matrix molecules. This study examines the effect of one PEMF upon the extracellular matrix and calcification of endochondral ossification in vivo. The synthesis of cartilage molecules is enhanced by PEMF, and subsequent endochondral calcification is stimulated. Histomorphometric studies indicate that the maturation of bone trabeculae is also promoted by PEMF stimulation. These results indicate that a specific PEMF can change the composition of cartilage extracellular matrix in vivo and raises the possibility that the effects on other processes of endochondral ossification (e.g., fracture healing and growth plates) may occur through a similar mechanism.
Serum albumin and its acid hydrolysis peptides dominate preparations of mineral-bound enamel proteins
Authors: Dr. Limeback Hardy , Sakarya Harry , Chu Willa , Mackinnon Mary
Published: February 2010

Serum albumin is a major noncollagenous protein component of bone, dentine, and, according to our results, enamel. Preparations of mineral-bound proteins from porcine developing enamel contain a single 67 kD protein at neutral pH or three proteins (67, 63, and 53 kD) at low pH that were assumed to be enamelins, a minor class of enamel proteins.(15) A more complete analysis of these proteins in this study showed that they were derived from porcine serum albumin (PSA). This was demonstrated by amino acid analysis, by N-terminal sequence analysis, by immunoblot studies using an anti-PSA antibody, and by SDS-PAGE analysis of the acid hydrolysis, cyanogen bromide, and tryptic peptides. Examination of enamel at different developmental stages showed that PSA deposited in enamel from the enamel organ and from the dentine during development, not during the dissection process. These results indicate that true enamelins must represent a very small fraction of the total mineral-bound protein matrix in porcine developing teeth, and this has important implications on the role of mineral-bound proteins during mineralization of enamel.
Analysis of parathyroid hormone in bovine parathyroid cysts
Authors: Ayer Linda M. , Szarka Roderick J. , Mortimer Shane T. , Alexander Fred J. , Martin Justin M. E. , Andersen Melvin A. , Dr. Hanley David A.
Published: February 2010

Parathyroid cysts were found in 2–5% of beef steers and heifers while collecting parathyroids at a local abattoir. The cyst fluid contained parathyroid hormone (PTH), averaging 15 ng/ml of fluid. Reversed-phase high-performance liquid chromatographic (HPLC) analysis of the fluid using an acetonitrile-trifluoroacetic acid solvent system, followed by carboxyl-terminal specific PTH radioimmunoassay of the eluant fractions, revealed two major peaks of PTH immunoreactivity. The first eluted at approximately 25% acetonitrile, a position similar to the commercially available human carboxyl terminal fragment of PTH-(39–84). The second peak, at approximately 37% acetonitrile, corresponded to the elution position for bovine PTH-(1–84). The cyst fluid was tested for enzyme activity by incubation of 125I-labeled bovine PTH with cyst fluid for 30 minutes at 37°C, which resulted in no fragmentation of the labeled peptide. Immunohistochemistry of the cells lining the parathyroid cyst indicated that they contained PTH.
Effects of altered calcium intake on diurnal and calcium-stimulated plasma calcitonin in normal women
Authors: Tiegs Robert D. , Heath Hunter
Published: February 2010

We sought to determine if any protective effect of dietary calcium (Ca) or Ca supplements on bone could be at least partially mediated by increased calcitonin (CT) secretion. First we studied 10 healthy premenopausal women (median age, 35.5 years) who were randomized to high or low dietary Ca intake (1752 versus 391 mg elemental Ca per day) for 2 weeks and then crossed over. At the end of each dietary period, blood was drawn on 1 day at 0800, 1200, 1700, and 2000 h to assess diurnal variation of plasma CT levels. CT secretory reserve was assessed on the next day by Ca infusion (2 mg Ca per kg body weight over 5 minutes). Next, we studied 10 healthy premenopausal women who took a low-Ca diet (∼ 400 mg Ca per day) for a 2 week control period. The women were then randomized to high- or low-Ca intake [400 mg dietary Ca ± 1500 mg Ca per day (as supplemental CaCO3)] and then crossed over. At the end of each study period, the diurnal variation in CT was tested on day 1; the CT secretory reserve was assessed on day 3 by an oral Ca load (500 mg as CaCO3)] and on day 5 by Ca infusion. Plasma immunoreactive CT was measured in whole plasma (iCT) and after silica extraction (exCT), predominantly monomeric CT. Neither increased dietary Ca nor Ca supplements affected the diurnal levels of iCT or exCT or augmented plasma CT responses to an oral Ca load. Furthermore, there was no change in secretory reserve for iCT or exCT after Ca infusion. We conclude that short-term Ca administration, in the form of dietary Ca or Ca salts, at the extremes of normal dietary intake and in doses normally used to treat osteoporosis, respectively, does not affect CT secretion in healthy premenopausal women. It seems unlikely that any potential beneficial effects of ingested Ca on the skeleton in women are mediated by CT.
Parathyroid hormone-related protein relaxes rat gastric smooth muscle and shows cross-desensitization with parathyroid hormone
Authors: Mok Lester L. S. , Ajiwe Eberechukwu , Martin T. John , Thompson James C. , Cooper Cary W.
Published: February 2010

Previously, we reported that parathyroid hormone (PTH) is a potent and effective relaxant of rat gastrointestinal smooth muscle. Since the recently discovered PTH-related protein (PTHrP) has amino-terminal homology with PTH and acts like PTH on bone and kidney, we decided to study the effects of synthetic PTHrP analogs on the isometric tension of rat fundic strips. Rat (r) PTH-(1–34), human (h) PTHrP-(1–34), and [Tyr0]hPTHrP-(1–34) relaxed acetylcholine-stimulated fundic strips in a dose-dependent manner, with an IC50 of 6, 10, and 31 nM, respectively. However, maximal doses of [Tyr0]hPTHrP-(1–34) were considerably less effective than the other two peptides. Addition of rPTH-(1–34) or hPTHrP-(1–34) to a maximally effective dose of [Tyr0]hPTHrP-(1–34) produced no further relaxation, indicating that [Tyr0]hPTHrP-(1–34) also has antagonistic properties. Bovine PTH-(3–34), an established in vitro antagonist of PTH, partially inhibited the relaxant effect of PTHrP. Fundic strips that had been desensitized by preincubation for 30–45 minutes with either rPTH-(1–34) or hPTHrP-(1–34) (330–500 nM) were also insensitive to the relaxant action of either peptide, but in the same preparations, the relaxation produced by vasoactive intestinal peptide was unaffected. These studies indicate that PTHrP and PTH can interact with the same receptor. Whether PTHrP influences gastrointestinal motility in normal or tumor-bearing persons remains to be investigated.
Muscle strength, physical fitness, and weight but not age predict femoral neck bone mass
Authors: Pocock Nicholas , Dr. Eisman John , Gwinn Tom , Sambrook Philip , Kelly Paul , Freund Judith , Yeates Michael
Published: February 2010

Hip fractures are the most serious complication of osteoporosis. Although low proximal femoral bone mineral density (BMD) does not cause hip fractures directly, it is clearly a prerequisite for the increased risk associated with aging. To investigate the mechanism of the age-related decline in proximal femoral bone mineral density, we have examined the relative importance of muscle strength, physical fitness, and body mass index (BMI) in addition to age in the determination of proximal femoral BMD in 73 healthy female volunteers age 20–75 years.

Muscle strength was an independent predictor of BMD at all three sites in the proximal femur as well as in the lumbar spine and forearm; proximal femur BMD was also predicted by physical fitness. BMI was a positive predictor of bone mass at all sites. In the proximal femur, age was not an independent predictor of BMD at any site.

In postmenopausal women muscle strength was a significant predictor of bone mass in the femur and forearm, but not in the spine. However, BMI remained predictive of bone mineral at all sites.

Muscle strength, physical fitness, and weight appear to exert independent effects upon bone mass. Age effects may be mediated indirectly through associated changes in these factors. The integrated physical load on the skeleton may be a final common pathway.
Resorption is not essential for the stimulation of bone growth by hPTH-(1-34) in rats in vivo
Authors: Dr. Hock J. M. , Hummert J. R. , Boyce R. , Fonseca J. , Raisz L. G.
Published: February 2010

Chronic low doses of hPTH-(1–34) stimulate bone growth in rats in vivo. The objective of these studies was to determine if the anabolic effect of hPTH-(1–34) on rat bone in vivo is dependent on an initial stimulation of resorption by blocking resorption with either salmon calcitonin (CT) or dichloromethylene diphosphonate (Cl2MDP).

Male Sprague-Dawley rats, 70–100 g, were treated with daily subcutaneous (SC) injections of vehicle (V) or hPTH-(1–34), 8 μg per 100 g (PTH), for 12 days. In experiment 1, rats were given CT for 3 (CT3) or 12 (CT12) days, either alone or in combination with hPTH-(1–34) (CT3-PTH and CT12-PTH) or vehicle for 12 days. In experiment 2, rats were pretreated for 4 days with Cl2MDP or its vehicle before starting the daily PTH or vehicle injections. Rats were then killed. Sera, femora, tibiae, and kidneys were removed for chemical and histomorphometric analyses.

PTH, PTH-CT3, and PTH-CT12 rats showed significant increases in total bone calcium (18–23%), dry weight (DW, 13–25%), and bone-forming surfaces compared with their respective controls. Eroded (resorption) surfaces were comparable between the groups. Although weight gain and serum calcium were normal in rats treated for 3 days with CT, rats treated for 12 days with CT gained 14% less weight than controls and were hypophosphatemic, with reduced serum calcium and urea nitrogen. Total bone mass increased both in Cl2MDP rats (Ca 21%, DW 2%), where resorption was presumably blocked, and in PTH rats (Ca 31%, DW 19%). The increase in bone mass was greater in PTH-Cl2MDP rats (Ca 48%, DW 29%) than in rats treated with Cl2MDP alone, suggesting that although Cl2MDP blocked resorption, the anabolic response to PTH was not altered. As neither short-term treatment with CT nor Cl2MDP blocked the anabolic response of bone to hPTH-(1–34), this response does not appear to depend on the early stimulation of resorption.
Evaluation of the osteoclastic population in iliac crest biopsies from 36 normal subjects: A histoenzymologic and histomorphometric study
Authors: Dr. Palle S. , Chappard D. , Vico L. , Riffat G. , Alexandre C.
Published: February 2010

After histochemical staining of tartrate-resistant acid phosphatase (TRAP) activity, the total and active trabecular resorption surfaces and the number of osteoclasts were determined by histomorphometry on iliac crest biopsies from 36 healthy volunteers. The subjects were separated into three groups according to age and sex. Total trabecular resorption surface showed no significant variation in any group, but the fraction of active resorption surface was significantly higher in the older population. The number of TRAP cells per mm2 of section area, related to trabecular bone volume or surface, showed a significant increase in elderly subjects. The mean osteoclast interface was similar in all the groups. We found a significant decrease in resorption depth between young and old populations. These results are consistent with a reduced activity of bone-resorbing cells in advancing age. These normal values, established after histochemical identification of osteoclasts, may be applied for evaluating abnormal bone-resorbing cell activity in metabolic bone diseases.
Effect of long-distance running on bone mass in women
Authors: Kirk Shelley , Sharp Charles F. , Elbaum Nancy , Endres D.B. , Mohler John G. , Rude Robert K. , Simons Steven M.
Published: February 2010

The effect of long-distance running on bone mass was assessed in 10 premenopausal and 9 estrogen-deprived postmenopausal women and compared to that in closely matched sedentary control women. Vertebral trabecular bone density (VBD) was determined by computed tomography and radial cortical bone density (CBD) by single-photon absorptiometry. Physical fitness was assessed by determining maximal oxygen consumption (VO2max) on a treadmill run to exhaustion. VBD was 183 + 7 mg/cm3 and VO2max was 48 + 1 ml/kg per minute in young women runners and 163 + 8 mg/cm3 and 32 + 2 ml/kg per minute in sedentary young women. A positive correlation was noted between VBD and VO2max in these groups (r = 0.509, p < 0.03). Despite a significantly higher VO2max in postmenopausal women runners compared with sedentary controls (37 + 2 versus 24 + 2 ml/kg per minute), VBD was identical (112 + 5 versus 111 + 5 mg/cm3) and no correlation was seen between VBD and VO2max (r = 0.187, p = 0.457). Radial cortical bone density was not different between the runners or sedentary groups in young women (0.738 + 0.01 versus 0.732 + 0.1 g/cm2) or postmenopausal women (0.617 + 0.3 versus 0.665 + 0.4 g/cm2). These results suggest that although physical fitness enhances vertebral bone density in premenopausal women, it does not appear to prevent age-and/or sex steroid deficiency-induced bone loss in postmenopausal women.
Articular chondrocytes lose their proliferative activity with aging yet can be restimulated by PTH-(1–84), PGE1, and Dexamethasone
Authors: Livne Erella , Weiss Anna , Dr. Silbermann Michael
Published: February 2010

Mouse mandibular condyles develop spontaneous degenerative changes by 6 months of age, hence providing a good in vivo model for studies related to processes associated with the onset and progression of age-related osteoarthritis. Further, this joint provides an appropriate system to investigate the potential of articular cartilage to respond to hormones and local growth factors in old age. The present study examined (1) the age-related changes in [3H]thymidine incorporation by articular chondrocytes in the mouse mandibular condyle, and (2) the effect of systemic and local factors upon the tissue's ability to resume DNA synthesis. Condyles of female CW-1 mice ranging from 3 to 18 months of age were cultured in the presence of PTH-(1–84) (2 μ/g/ml), PGE1 (20 μ/ml), dexamethasone (10−7 M), and MSA (5 μg/ml) and were concomitantly labeled with [3H]thymidine. Autoradiographs were analyzed quantitatively and revealed (1) a significant (p < 0.01) age-related decrease (–80%) in the labeling index of the articular cartilage, and (2) the ability of old tissues to resume DNA synthesis following in vitro treatment with PTH-(1–84), PGE1, and dexamethasone. Concomitant quantitative incorporation studies further substantiated the autoradiographic findings. Hence, these factors possess a direct stimulatory effect upon senescent chondrocytes involved in an advanced stage of spontaneous osteoarthritis.
Interferon γ and calcitonin induce differential changes in cellular kinetics and morphology of osteoclasts in cultured neonatal mouse calvaria
Authors: Klaushofer Klaus , Hoffmann Oskar , Czerwenka Erwin , Koller Kristian , Hörandner Helmut , König Uwe , Peterlik Meinrad
Published: February 2010

The present study compares the effects of calcitonin (CT) and interferon γ (IFN-γ) on the size, distribution, and ultrastructure of osteoclasts in cultured neonatal mouse calvaria. The number and cross-sectional area of osteoclasts in cultured bones was increased by the addition of parathyroid hormone (PTH) to the culture medium for 24–48 h. Prolonged treatment (up to 72 h) with PTH led to extensive rarefication and formation of holes in the mineralized matrix. PTH-activated osteoclasts exhibited an elaborate ruffled border and showed a typical zonal arrangement of intracellular organelles with the outer cytoplasmic region containing numerous membrane-bound vesicles. CT (20 mU/ml) within 90 minutes caused a complete loss of the ruffled border in PTH-activated osteoclasts. The typical zonal architecture disappeared with intracellular vesicles spread through the entire cytoplasm. Prolongation of CT treatment to 24–48 h led to the appearance of vesicles with dark granular content in inactivated osteoclasts. This morphologically distinct vesicle population allowed us to identify the latter cells as “postosteoclasts” and thereby to distinguish them from osteoclasts precursors. Small active osteoclasts that had originated very likely from these precursors appeared in calvarial bones coincidentally with escape from CT inhibition of bone resorption and resulted in increased total number of osteoclasts. In contrast, IFN-γ (500 U/ml) acting as a proliferation inhibitor, reduced the total number of osteoclasts. In the presence of PTH it caused no immediate (90 minutes) change in the ultrastructure of PTH-induced osteoclasts apart from an increase in the number of autophagic vacuoles. After prolonged exposure (24–48 h) in the presence of PTH, osteoclasts with low resorbing activity exhibited intermediate borders at their contact zone with the mineralized matrix. In the absence of PTH, the short-term effect of the immune interferon on osteoclast morphology was almost comparable to that of CT. After prolonged treatment in the absence of PTH, postosteoclasts comparable in vesicle population and size to those after CT treatment were found.
Identification and characterization of parathyroid hormone receptors in rat renal cortical plasma membranes using radioligand binding
Authors: Nickols G. Allen , Metz-Nickols Maureen A. , Roberts M. Scot , Cooper Cary W. , Pang Peter K.T.
Published: February 2010

Parathyroid hormone (PTH) receptors have been described in renal tissue from several species, but not in the rat. In this study, radioligand binding techniques were used to identify and characterize PTH receptors in rat kidney cortical membranes. The sulfur-free PTH analog [Nle8,18Tyr34]bovine PTH-(1–34)amide was iodinated using the iodogen method. This ligand was suitable for use in identifying PTH receptors in canine renal membranes, but not rat renal membranes. Synthetic, unsubstituted rat PTH-(1–34) was iodinated using the milder, lactoperoxidase technique and was purified by HPLC on a C8 column. [125I]rat PTH-(1–34) bound rapidly to both rat and dog renal membranes. At 22°C the reaction reached steady state within 20 minutes, and this level was maintained for at least 3 h. Specific binding was routinely >90% for rat kidney and >95% for dog kidney. Similar results were obtained at 4°C with a longer time required to attain steady state (approximately 45 minutes). Binding was reversible as demonstrated by dissociation of bound ligand after either infinite dilution or displacement with excess nonradioactive PTH. Binding was saturable and of high affinity (rat kidney: Bmax = 2.3 pmol/mg protein, Kd = 3.1 nM, dog kidney: Bmax = 2.1 pmol/mg protein, Kd = 3.7 nM). Rat renal cortical adenylate cyclase activity was stimulated by rat PTH in a dose-dependent manner with an EC50 of 4 nM, a value in good agreement with the binding data. This study demonstrates the feasibility of identifying and characterizing parathyroid hormone receptors in rat renal cortical plasma membranes using radioligand binding techniques.
Estrogen receptors and human bone cells: Immunocytochemical studies
Authors: Colston Kay W. , King R.J.B. , Hayward J. , Fraser D.I. , Horton M.A. , Stevenson J.C. , Arnett T.R.
Published: February 2010

In this immunocytochemical study we have probed a number of human bone cell types and bone preparations for the presence of the estrogen receptor (ER) with two distinct monoclonal antibodies. Using a well-validated antibody (H222) that recognizes human ER and standard peroxidase-antiperoxidase methodology, we were unable to demonstrate nuclear staining for ER in cultured primary or transformed human bone-derived cells or in fetal bone sections. Attempts to visualize ER in osteosarcoma cell lines (TE85C and HTB96) using a silver enhancement procedure were also unsuccessful. Additionally, we failed to detect immunocytochemical staining for the progesterone receptor (using monoclonal antibody mPR1) in control or estrogen-treated human bone cell cultures. Estrogen and progesterone receptor staining was readily detectable in MCF7 human breast cancer cells. In contrast, with a monoclonal antibody that recognizes a 29 kDa cytoplasmic component (p29) closely related to human ER, we observed specific staining in all the osteoblastlike cells studied. Cytoplasmic staining for this p29 antigen was most intense in primary cultures of human bone-derived cells. It is possible that the relatively abundant but as yet undefined p29 antigen may act as a sensitive marker for the presence of ER in cells at levels below the detection limit of the anti-ER monoclonal antibody. If so, our results are consistent with the presence of ER in osteoblastlike cells at very low concentrations.
Biomechanical properties of the proximal femur determined in vitro by single-energy quantitative computed tomography
Authors: Esses Steven I. , Lotz Jeffrey C. , Hayes Wilson C.
Published: February 2010

To assess the use of quantitative computed tomography as an in vivo predictor of fracture in the osteoporotic hip, we examined the in vitro relationship between single-energy quantitative computed tomography data, calibrated for scanner drift, and the mechanical properties of trabecular bone from the proximal femur. For 49 samples, the apparent density and ultimate strength were measured and their functional relationship to the computed tomography data determined. Apparent density demonstrated a moderate linear correlation to the computed tomography numbers (R2 = 0.60), and the ultimate strength was related through a power law (R2 = 0.83). In addition, for 8 intact femora, average computed tomographic data from the sub-capital region were moderately correlated to the ultimate fracture load applied under controlled in vitro conditions (R2 = 0.64). The average fracture energy for these femora was 43 J, a value more than an order of magnitude less than the energy available in a fall from standing height, suggesting that fall mechanics are a more important determinant of fracture risk than has been previously thought. The relationship between the energy absorbed to failure and the computed tomography data was best described by a power law (R2 = 0.90). Based on these results, it appears that quantitative computed tomography provides a potentially useful approach for the direct estimate of that component of fracture risk that can be attributed to a reduction in bone strength.
High extracellular calcium increases the production of a parathyroid hormone-like activity by cultured leydig tumor cells associated with humoral hypercalcemia
Authors: Dr. Rizzoli R. , Bonjour J.-P.
Published: February 2010

Cultured Leydig tumor cells produce a parathyroid hormone (PTH)-like activity, but little is known about the regulation of the release of this factor. In the present work, we investigated the influence of the extracellular calcium concentration on the production of adenylate cyclase-stimulating activity, as evaluated in the osteoblast-like PTH-responsive cell line UMR 106. Medium conditioned in the presence of 0.4 mM or 3 mM Ca elicited a 5.8 ± 0.4-fold and 10.3 ± 0.9-fold increase over basal of cAMP production, respectively (p < 0.001, n = 11 experiments). This effect, which was selective for PTH-like activity, was detectable after 2 h of incubation and maximal at 6–14 h. It was abolished by the protein synthesis inhibitor cycloheximide, but not by actinomycin D or cordycepin, suggesting a post-transcriptional site of action. Thus, the production of a tumoral circulating factor implicated in the pathogenesis of humoral malignant hypercalcemia may be influenced in a positive way by an increase in extracellular calcium concentration.
Central osteosclerosis with ectodermal dysplasia: Clinical, laboratory, radiologic, and histopathologic characterization with review of the literature
Authors: Civitelli Roberto , McAlister William H. , Teitelbaum Steven L. , Dr. Whyte Michael P.
Published: February 2010

IBIDS is a syndrome characterized by ichthyosis, brittle hair, impaired intelligence, decreased fertility, and short stature, but unassociated with skeletal lesions. This condition is considered a form of trichothiodys-trophy because hair from several cases has been found to have a low sulfur content. We describe a 9-year and 10-month-old white boy whose clinical features resemble the IBIDS syndrome (ichthyosis, brittle hair, cataracts, and short stature), but who also has marked axial osteosclerosis and peripheral osteopenia. No abnormalities of mineral homeostasis were noted. Histopathologic assessment of nondecalcified bone specimens excluded osteopetrosis, but suggested slow skeletal remodeling. When subjected to polarized light microscopy, his hair exhibited the band-like pattern of birefringence described in trichothiodystrophy. Literature review disclosed 8 patients, 2 of whom had been diagnosed as trichothiodystrophy, with like clinical features including osteosclerosis. These skeletal abnormalities together with clinical features of the IBIDS/trichiodystrophy syndrome, we believe, reflect the prototype of a disorder that seems best described as central osteosclerosis with ectodermal dysplasia.