bluuebunny/crossref_metadata_2025 huggingface dataset:
Sex differences in peak adult bone mineral density
Authors: Kelly Paul J. , Twomey Lance , Sambrook Philip N. , Dr. Eisman John A.
Published: February 2010

Osteoporotic fractures are more common in women than men. Although accelerated bone loss following the menopause is recognized as of major importance, it is generally considered that a lower peak adult bone mass in females also contributes to their increased risk of osteoporosis in later life. To examine potential sex differences in peak adult bone mass we studied 29 pairs of dizygotic twins of differing within-pair sex in whom the female twin was premenopausal (mean age 37 years, range 21–55). Bone mineral density (BMD, g/cm2) was measured at the lumbar spine and femoral neck by dual-photon absorptiometry; 22 pairs also had BMD measured in the distal and 21 pairs in the ultradistal radius by single-photon absorptiometry. There was no significant difference in usual dietary calcium intake or tobacco consumption between the twin pairs. Consistent with accepted dogma, BMD at both radial sites were higher (+27%) in the males than their female cotwins. In contrast, there was no sex difference (male versus female) in BMD (mean ± SEM) in the femoral neck (0.96 ± 0.02 versus 0.97 ± 0.03), and surprisingly, the females had a greater lumbar spine BMD than their male cotwins (1.19 ± 0.03 versus 1.26 ± 0.03, p < 0.05). This difference was observed despite the fact that the males were taller (p = 0.033). If the femoral neck BMD values in the females were corrected for this difference in BMI, their values (0.99 ± 0.03 g/cm2) were significantly higher than those in their male cotwin (p < 0.05). Alternatively if the lumbar spine BMD in the female twins was corrected for the sex difference in anterior-posterior vertebral dimension (measured in cadaveric vertebrae) the female values were then 15% greater than those of their male cotwin. Therefore, a sex difference in peak adult bone density at the lumbar spine or femoral neck does not contribute to the higher incidence of osteoporotic fracture at these sites in aging women. Rather, the data suggest that before the menopause the female sex is associated with a bone density greater than expected on the basis of muscle strength or body size or weight.
Glucocorticoids stimulate resorption in fetal rat parietal bones in vitro
Authors: Dr. Gronowicz Gloria , McCarthy Mary Beth , Raisz Lawrence G.
Published: February 2010

The effect of glucocorticoids on bone resorption was examined in a serum-free mineralizing organ culture system derived from 20 day fetal rat parietal bones. Bone resorption was assessed by prelabeling the fetal rats in utero with 45Ca and determining the daily release of 45Ca into the medium of cultured bones. During the first 24 h of treatment a transient stimulation of bone resorption was found; 4.5 ± 0.3% of the total 45Ca was released into the medium with 1 nM corticosterone and 4.1 ± 0.2% with 10 nM corticosterone compared to 2.9 ± 0.2% in control bones. Treatment with 1 and 10 nM dexamethasone for 24 h also showed an increase in 45Ca release compared to control bones. During the same time period 45Ca release was 6.9 ± 1.4% with 10 nM parathyroid hormone. At later time points 100 and 1000 nM corticosterone inhibited 45Ca release, but 1 and 10 nM corticosterone values were similar to controls. At 24 h the number of osteoclasts per mm2 tissue in bone lacunae was significantly elevated with 1–100 nM corticosterone and 10 nM parathyroid hormone compared to control bones. In control bones 0.10 ± 0.05 osteoclasts per mm2 of tissue were found, but 0.59 ± 0.21 osteoclasts per mm2 were seen with 10 nM corticosterone and 1.50 ± 0.34 with 10 nM parathyroid hormone. An additional assay of bone resorption, the release of lysosomal β-glucuronidase into the medium was also elevated in glucocorticoid and parathyroid hormone-treated cultures. During 0–24 h, the medium from 10 nM parathyroid hormone-treated bones contained elevated levels of β-glucuronidase activity, 7.5 ± 0.7 versus 6.0 ± 0.4 μg/ml in the control bones and, during 24–48 h, 10.5 ± 0.8 versus 7.7 ± 0.6 μg/ml of phenolphthalein in the controls. With 10 nM corticosterone there was a significant increase of 10.0 ± 0.5 μg/ml released at 24–48 h, but high concentrations of glucocorticoids decreased β-glucuronidase release. We conclude that corticosterone can cause a transient increase in bone resorption by increasing osteoclast number and activity in bone.
Intraperitoneal free fatty acids induce severe hypocalcemia in rats: A model for the hypocalcemia of pancreatitis
Authors: Dettelbach Mark A. , Deftos Leonard J. , Stewart Andrew F.
Published: February 2010

Indirect evidence suggests a causative role for intraperitoneal free fatty acids (FFA) in hypocalcemia associated with pancreatitis. We examined the effects of intraperitoneal injection of four naturally occurring FFAs on serum calcium in rats. Two saturated FFAs, stearate and palmitate, induced little or no hypocalcemia. Two unsaturated FFAs, oleate and linoleate, caused dramatic hypocalcemia in treated versus control rats (6.3 ± 1.4 and 5.3 ± 0.7 mg/dI, respectively, versus 10.1 ± 0.6). Dose-response studies demonstrated that minute quantities of oleate (100 μl per 250 g rat) caused marked hypocalcemia (7.2 ± 0.3 mg/dI). Treated versus control rats also revealed a decrease in ionized calcium (3.15 ± 0.2 versus 5.6 ± 0.05 mg/dI) and magnesium (1.4 ± 0.15 versus 2.0 ± 0.10), an appropriate increase in PTH levels (1670 ± 451 versus 396 ± 235 pg/ml), and a fall in calcitonin levels (70.4 ± 21.3 versus 47.5 ± 16.4 pg/ml) but no change in albumin or phosphate levels. In vitro, the Ksp of calcium dioleate was shown to be 5.3 × 10-−8 m3/liter3; thus under physiologic conditions 100 μl oleate binds 7.2 mg calcium, or approximately twice the total ECF ionized calcium in the rat.

The amounts of intraperitoneal FFA that can easily be achieved in pancreatitis complex pathophysiologically significant amounts of calcium and may lead to severe hypocalcemia.
Case report: Idiopathic acquired diffuse osteosclerosis in a young woman
Authors: Beyer H. Stephen , Parfitt A. Michael , Shih Mei Shu , Anderson Quentin , Heath Hunter
Published: February 2010

We describe a young woman who acquired a painful, diffuse osteosclerosis of the cervical, thoracic, and lumbar spine, pelvis, and long bones of the legs as an adult. Bone densitometry showed a large increase in apparent bone density. Skeletal radiographs demonstrated progressive endosteal and periosteal thickening of the cortices. A bone scan showed increased uptake of radiolabel. The serum total alkaline phosphatase and 1,25-(OH)2D3 levels were substantially elevated and the immunoreactive PTH was mildly elevated. Bone biopsy showed increased bone turnover, especially on endocortical and intracortical surfaces, but the structural indices were normal. By 4 years after presentation the bone pain had remitted and the serum alkaline phosphatase, 1,25-(OH)2D3, and PTH were normal. No cause for the occurrence of osteosclerosis in this patient could be found.
Rapid publication: Constitutive expression of a vitamin D 1-hydroxylase in a myelomonocytic cell line: A model for studying 1,25-dihydroxyvitamin D production in vitro
Authors: Dr. Adams John S. , Beeker Timothy G. , Hongo Takashi , Clemens Thomas L.
Published: February 2010

The capacity of the v-myc-transformed, chicken myelomonocytic cell line HD-11 to metabolize 25-hydroxyvitamin D3 (25-OHD3) was examined. HD-11 cells produced and secreted a metabolite of 25-OHD, that was bound with high affinity by receptor for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. On normal-phase HPLC, this metabolite cochromatographed with authentic 1,25-(OH)2D3 in both hexane- and methylene chloride-based solvent systems. The 25-OHD, 1-hydroxylation reaction was substrate saturable with a Km of 73 nM 25-OHD3 and a maximal velocity of 167 fmol per 106 cells per h. This reaction was inhibited by keto-conazole, a recognized inhibitor of cytochrome P450 mixed-function oxidases including the authentic, renal 25-OHD3 1-hydroxylase. On the other hand, HD-11 cell 1,25-(OH)2D3 production was not affected by the antioxidant DPPD, a known inhibitor of free radical-generated 1,25-(OH)2D3. In addition to synthesizing 1,25-(OH)2D3, this monocyte-macrophage cell line also has the potential to be a target for the hormone; HD-11 cells express high-affinity receptor for 1,25-(OH)2D3 (Kin = 0.06 nM).

Division of Endocrinology and Metabolism, Cedars-Sinai Medical Center-UCLA School of Medicine, Los Angeles, CA 90048.
A comparison of morphometric definitions of vertebral fracture
Authors: Smith-Bindman Rebecca , Cummings Steven R. , Steiger Peter , Genant Harry K.
Published: February 2010

To compare the accuracy of several approaches for defining prevalent vertebral fractures from measurements of vertebral dimensions (morphometry), we measured the lateral dimensions of vertebral bodies of 115 normal premenopausal and 100 postmenopausal women. Of the postmenopausal women two observers agreed that 49 had definite vertebral fractures and 38 were definitely normal. Using these classifications as an independent reference, women were then classified as fractured or normal by several definitions based on vertebral morphometry. No morphometric definition of vertebral fracture agreed perfectly with the consensus classifications. In general, definitions that involved combinations of measurements of anterior (Ha), middle (Hm), and posterior (Hp) vertebral height classified women more accurately than did definitions based on a single measurement or ratio. The Ha/Hp ratio produced many false positives unless it was adjusted for normal variations in the shapes of different vertebral bodies. Definitions of fracture based on a > 15% reduction in heights or ratios had higher sensitivity but more false positives than definitions that used a more stringent (>20%) criterion. All morphometric definitions of vertebral fracture separated the postmenopausal women into two groups (fractured and normal) that had significantly (P < 0.001) different mean spine bone density by quantitative computed tomography. Definitions that had the lowest rates of false positives also produced the largest differences in bone density between those defined as fractured and those defined as normal.
Noninvasive assessment of ulnar bending stiffness in women
Authors: McCabe Frances , Zhou L.-J. , Steele Charles R. , Marcus Robert
Published: February 2010

The load-carrying capacity of cortical bone is closely related to its geometry and to its fundamental material properties, including mineral content (BMC). Together these determine the bending stiffness EI, where I is the cross-sectional moment of inertia and E is Young's modulus of elasticity. To assess the relationship of BMC and bone width (BW) to EI in healthy women, we used mechanical response tissue analysis (MRTA), a noninvasive method that involves analysis of tissue responses to ulnar vibration. A total of 48 healthy women were enrolled into an older (64 ± 1y, n = 25) and a younger (25 ± 0.6y, n = 23) group. BMC and BW of the dominant ulna were measured by single-photon absorptiometry (SPA). EI was determined by MRTA. BMC (0.75 ± 0.02 versus 0.63 ± 0.02 g/cm), BMC/BW (0.75 ± 0.02 versus 0.63 ± 0.02 g/cm2), and EI (21.7 ± 1.3 versus 21.3 ± 1.1 N ṁ m2) were significantly greater (p < 0.005) in the young subjects. BW did not change with age (1.00 ± 0.01 versus 1.01 ± 0.01 cm). In young women, simple correlations of BMC and BW with EI were both significant. By multiple regression analysis only BW independently predicted EI (EI = −0.35 + 39.1 × BMC, R2 = 0.52). In older women BMC and BW correlated with EI, but in multiple regression only BMC was significant (EI = −34.5 + 62.1 × BW; R2 = 0.45). When this analysis of older women included only those whose BMC values were within 2 SD of the young mean, BMC remained the only significant predictor of EI. We conclude that ulnar dimensions make important contributions to axial loading strength in healthy young women, whereas changes in internal architecture, porosity, or bone material properties increase the importance of BMC as a determinant of ulnar stiffness for older women.
Urinary excretion of parathyroid hormone-related protein fragments in patients with humoral hypercalcemia of malignancy and hypercalcemic tumor-bearing nude mice
Authors: Imamura Hidehito , Dr. Sato Kanji , Shizume Kazuo , Satoh Tomoko , Kasono Keizo , Ozawa Minoru , Ohmura Eiji , Tsushima Toshio , Demura Hiroshi
Published: February 2010

To investigate whether parathyroid hormone-related protein (PTHrP), a hypercalcemia-inducing factor responsible for malignancy-associated hypercalcemia (MAH), is excreted into urine of these patients, radioimmunoassay was established using antiserum specific for the C-terminal region of PTHrP-(127–141).

Immunoreactive PTHrP (iPTHrP) was detected in the urine of all patients with MAH (n = 6) in whom nephrogenous cyclic AMP excretion was elevated. However, iPTHrP was not detected in the urine of normal subjects (n = 25) or hypercalcemic patients with primary hyperparathyroidism (n = 8). In normocalcemic patients with malignant disorders iPTHrP was not detected in the urine in most cases (24 of 25 patients) but was detectable in 1 of 25 patients. iPTHrP was also detected in the urine of hypercalcemic nude mice transplanted with PTHrP-producing tumors, but not in the urine of control and normocalcemic nude mice transplanted with PTHrP-nonproducing tumor. Furthermore, size-exclusion high-performance liquid chromatography revealed that the molecular weight of iPTHrP is about 2000–6000 daltons in the urine of patients as well as tumor-bearing nude mice.

These data indicate that the fragments of the C-terminal region of PTHrP are excreted into the urine of patients with MAH and in a few normocalcemic patients with malignancies, suggesting that the measurement of iPTHrP in the urine is potentially useful in the differential diagnosis of hypercalcemia, particularly in differentiating humoral hypercalcemia of malignancy and primary hyperparathyroidism.
Interrelationship among vitamin D metabolism, true calcium absorption, parathyroid function, and age in women: Evidence of an age-related intestinal resistance to 1,25-dihydroxyvitamin D action
Authors: Eastell Richard , Yergey Alfred L. , Vieira Nancy E. , Cedel Sandra L. , Kumar Rajiv , Dr. Riggs B. Lawrence
Published: February 2010

We studied the mechanism of impaired calcium absorption with aging in 51 healthy women whose ages ranged from 26 to 88 years. Serum concentrations of 1,25-dihydroxyvitamin D [1,25-(OH)2D, mean of four measurements per subject] increased with age by 22% (P < 0.05) but, by split-point analysis, plateaued or decreased slightly after age 65. In a subset of 20 subjects, [3H]1,25-(OH)2D3 kinetic analysis showed that this increase with age resulted from both increased production and decreased metabolic clearance of 1,25-(OH)2D. Despite the increase in serum 1,25-(OH)2D concentration, true calcium absorption did not change with age. The expected inverse correlation between true fractional calcium absorption and dietary calcium intake, however, was easily demonstrated (r = 0.66, P < 0.001). Serum intact parathyroid hormone (PTH) increased with age by 35% (P < 0.02) and serum bone gla protein (BGP, osteocalcin) increased by 47% (P < 0.001); the increases in serum PTH and serum BGP were directly correlated (r = 0.32, P < 0.05). The data are consistent with the following hypothetical model: (1) intestinal resistance to 1,25-(OH)2D action accounts for the increase in serum 1,25-(OH)2D concentrations with aging with no change in true calcium absorption; (2) this results in a compensatory increase in PTH secretion and in 1,25-(OH)2D production that prevents true calcium absorption from decreasing; (3) the previously described defect in 25-hydroxyvitamin D (25-OHD) 1α-hydroxylase activity in aging animals and humans acccounts for the leveling off in serum 1,25-(OH)2D concentration after age 65 years; and (4) the secondary hyperparathyroidism leads to increased bone turnover and thus contributes to age-related bone loss.
Osteotropic factor responsiveness of highly purified populations of early and late precursors for human multinucleated cells expressing the osteoclast phenotype
Authors: Kurihara Noriyoshi , Civin Curt , Roodman G. David
Published: February 2010

Recently we have adapted human long-term bone marrow cultures to form multinucleated cells (MNC) that express the osteoclast phenotype and used semisolid culture techniques to identify early (bipotent) and late (unipotent) mononuclear precursors for these MNC. The early precursor can form both osteoclast-like MNC and macrophage polykaryons; the late precursor forms only osteoclast-like MNC. In this study we examined the effects of osteotropic hormones and cytokines of MNC formation from highly purified populations of these early or late mononuclear precursor cells. MNC expressing the osteoclast phenotype were identified by their cross-reactivity with the 23c6 monoclonal antibody, which preferentially identifies osteoclasts. 1,25-(OH)2D3 (10−8 M), IL-1β (10 u/ml), and IL-6 (100 pg/ml) stimulated formation of 23c6-positive MNC from highly purified populations of early or late precursor cells. In contrast, PTH (50 ng/ml) did not act directly on late precursor cells but only stimulated 23c6-positive MNC formation from early precursors. These results show that (1) 1,25-(OH)2D3, IL-1β, and IL-6 can stimulate 23c6-positive MNC formation from a highly enriched population of early and late precursors, and (2) PTH does not act on late precursors but may act indirectly on the late precursors.
Comparison of dual-photon absorptiometry systems for total-body bone and soft tissue measurements: Dual-energy X-rays versus gadolinium 153
Authors: Russell-Aulet Mary , Wang Jack , Thornton John , Pierson Richard N.
Published: February 2010

A total of 81 subjects (41 males and 40 females) were scanned by dual-photon absorptiometry by 153Gd source (DPA; Lunar DP4) and by dual-energy x-ray absorptiometry (DEXA; Lunar-DPX) within a 24 h period. Total-body bone mineral density (TBMD), calcium content (Ca), and soft tissue mass (ST) were determined with a precision of about 1–1.5% using DPA and 0.5–1.0% using DEXA. Measurements of TBMD, Ca, ST, bone area (area), percentage fat, and regional bone mineral densities (BMD) were compared. Paired t-tests showed small but significant differences between all measurements. Correlations (r) for TBMD, Ca, area, ST, percentage fat, arm BMD, leg BMD, and trunk BMD were 0.99, 0.99, 0.97, 0.99, 0.97, 0.99, 0.99, and 0.98. There were small systematic differences for TBMD (<1%), calcium (3%), bone area (3%), soft tissue mass (7%), and percentage fat (9%) between the two approaches. Regression equations are given relating these measurements.
Acute effect of 1,25-dihydroxyvitamin D3, prednisone, and 1,25-dihydroxyvitamin D3 plus prednisone on serum osteocalcin in normal individuals
Authors: Nielsen Henning K. , Brixen Kim , Kassem Mustapha , Mosekilde Leif
Published: February 2010

Suppression of osteoblastic function plays an important pathogenic role for the development of glucocorticosteroid-induced osteoporosis. Serum osteocalcin (OC) is a sensitive marker of bone formation. The diurnal rhythm in serum OC can be changed by administration of single doses of either 1,25-(OH)2D3 or prednisone. However, the two steroids have opposing effects: 1,25-(OH)2D3 increases and prednisone decreases serum OC. The aim of the present study was to examine whether 1,25-(OH)2D3 can oppose the acute suppressive effect of prednisone on serum OC in normal subjects. We compared the effect of a combined dose of 2 μg 1,25-(OH)2D3 and 10 mg prednisone on the diurnal rhythm of serum OC with the effect of 2 μg 1,25-(OH)2D3 + placebo in a crossover study. Seven normal subjects aged 23–36 years were investigated twice at an interval of 1 week. Blood samples were collected every 60 minutes from 1900 until 1100 h the following day. Study drugs were given at 2000 h. The data from the present investigation were compared with data obtained from a similar study with placebo and prednisone in the same subjects. After administration of 1,25-(OH)2D3 serum OC followed the placebo curve during the first 8 h, but in contrast to the placebo curve it then continued to increase and remained elevated throughout the observation period (p < 0.05). Prednisone inhibited and reversed the nocturnal rise in serum OC levels (p < 0.01). The course of serum OC after administration of 1,25-(OH)2D3 + prednisone almost paralleled the course after placebo. We conclude that 1,25-(OH)2D3 and prednisone have opposing effects on serum OC.
Bradykinin induces formation of inositol phosphates and causes an increase in cytoplasmic Ca2+ in the osteoblastic cell line MC3T3-E1
Authors: Ljunggren Östen , Johansson Hans , Ljunghall Sverker , Fredholm Bertil B. , Lerner Ulf H.
Published: February 2010

Recordings of fura-2 fluorescence from single osteoblastic MC3T3-E1 cells showed that bradykinin (BK, 1 μM) induced a rapid increase in cytoplasmic free Ca2+ (Cai2+, from 114 + 13 to 239 + 17 nM, mean + SEM). Following this initial transient (<1 minute) increase there was a second slow increase in Cai2+ (from 117 + 11 to 151 + 12 nM). Incubation in buffer with no Ca2+ did not affect the first rapid BK-induced increase in Cai2+ but eliminated the second slow increase. Addition of indomethacin or hydrocortisone to the incubation buffer did not inhibit the effect of BK on Cai2+. BK caused a dose-dependent initial rapid increase in Cai2+ with threshold at 1 nM and a maximal effect (241 + 30% of basal Cai2+ concentration) at 0.1 μM. The B1 BK receptor agonist des-Arg9-BK (1 μM) caused only a small increase in Cai2+ in MC3T3-E1 cells (from 101 + 20 to 140 + 4 nM). BK dose and time dependently stimulated the formation of inositol phosphates in MC3T3-E1 cells with EC50 at 2.4 nM and a significant increase in inositol trisphosphate already seen after 15 s. The Ca2+ ionophore ionomycin induced a rapid increase in Cai2+ and prostaglandin E2 (PGE2) formation in MC3T3-E1 cells. Forskolin (10–30 μM) increased cyclic AMP accumulation but did not affect Cai2+ or PGE2 formation. Depletion of extracellular Ca2+ significantly reduced (but did not abolish) BK-induced PGE2 formation. The initial action of BK on Cai2+ is probably due to an inositol-(1,4,5)-trisphosphate-mediated rapid release of Ca2+ from intracellular stores in osteoblasts and is followed by an influx of extracellular Ca2+. The effect is due to B2 BK receptor occupancy and is not secondary to the prostaglandin synthesis. The BK-induced breakdown of phosphatidylinositol-(4,5)-bisphosphate with a subsequent increase in Cai2+ may be involved in BK-induced prostaglandin formation in osteoblasts.
Osteogenesis imperfecta: Changes in noncollagenous proteins in bone
Authors: Vetter U. , Fisher L.W. , Mintz K.P. , Kopp J.B. , Tuross N. , Termine J.D. , Robey P. Gehron
Published: February 2010

The noncollagenous proteins osteonectin, bone sialoprotein, osteocalcin, the small proteoglycan decorin (PG II), and α2-HS glycoprotein (which is synthesized in the liver but highly concentrated in bone) were measured in extracts of cortical bone from 3 type I, 2 type II, 8 type III and 13 type IV patients with osteogenesis imperfecta (OI) and from 7 control subjects.

Osteonectin was found to be reduced in the bone of all OI patients. The bone from severely affected type III OI patients contained the lowest levels of osteonectin. In contrast, bone sialoprotein was found to be elevated in the bones of OI patients. The highest levels were found in individuals classified as type IV patients. Osteocalcin and α2-HS glycoprotein concentrations were increased in all OI patients. Decorin levels were not significantly altered in OI bones compared to controls.

These changes in the concentrations of the noncollagenous proteins may contribute to the fragility of the OI bone by interfering with complete mineralization and/or normal tissue architecture.
Establishment and characterization of two immortalized cell lines of the osteoblastic lineage
Authors: Hofstetter W. , Guenther H.L. , Stutzer A. , Schenk R. , Fleisch H. , Friis R.
Published: February 2010

Osteoblastic cells were cloned by culturing rat calvariae cells in agarose in the presence of TGF-β and EGF. Two bone cell lines were established by immortalizing such an osteoblastic clonal cell population by the introduction of the avian v-mycOK10 gene in the form of a mouse ecotropic retrovirus. Although originating from the same clonal cell population, the two lines exhibited somewhat differing properties. IRC10/30-mycl expressed alkaline phosphatase (AP), showed PTH- and PGE2-induced cAMP production, synthesized mainly collagen type I and a minor fraction of type III, and produced mRNA for the bone-specific protein osteocalcin. IRC10/30-myc3 did not express AP, showed no PTH responsiveness, and synthesized only about one-third as much collagen as IRC10/30-mycl (4 versus 12% of total protein synthesis). However, the cell line IRC10/30-myc3 was induced to synthesize cAMP by PGE2 and produced osteocalcin mRNA. When cultured in vivo in diffusion chambers, both lines proved to be osteogenic. Besides bone, both lines also formed cartilage and fibrous tissue. Thus, by immortalizing a clonal cell population of the osteoblastic phenotype, cell lines expressing varying properties can emerge. Furthermore, the expression of alkaline phosphatase and PTH-inducible adenylate cyclase are not prerequisites for a cell to form bone in vivo. Finally, cells expressing the phenotype of differentiated osteoblasts, including osteocalcin synthesis, still have a multipotential differentiation capacity and form bone and cartilage in vivo.
Urinary excretion of pyridinoline crosslinks correlates with bone turnover measured on iliac crest biopsy in patients with vertebral osteoporosis
Authors: Dr. Delmas Pierre D. , Schlemmer Annette , Gineyts Evelyne , Riis Bente , Christiansen Claus
Published: February 2010

Vertebral osteoporosis, a common disorder in elderly women, is characterized by a wide spectrum of bone turnover abnormalities on iliac crest biopsy. The level of bone formation can be assessed noninvasively by measuring serum osteocalcin, whereas conventional biochemical markers of bone resorption lack specificity and do not reflect bone resorption assessed from histology. We measured the urinary excretion of pyridinoline crosslinks Pyr and D-Pyr, a specific marker of bone and cartilage collagen degradation, along with serum osteocalcin and urinary hydroxyproline, in 36 elderly women with vertebral osteoporosis who had a simultaneous iliac crest biopsy. Urinary pyridinoline crosslinks, but not hydroxyproline, correlated significantly with histologic resorption, assessed by the osteoclast surface (r = 0.35, p < 0.05 for Pyr; r = 0.46, p < 0.01 for D-Pyr). In addition, Pyr and D-Pyr were correlated with the bone formation rate as well as serum osteocalcin, with correlation coefficients ranging from 0.69 to 0.80, p < 0.0001. These data indicate that Pyr and D-Pyr are sensitive markers of bone turnover in elderly women with vertebral osteoporosis. The poor correlation between the level of urinary collagen crosslinks and histological assessment of bone resorption indicates the low sensitivity of iliac crest histomorphometry in the measurement of resorption rate of the skeleton.
Application of reflected light microscopy to identify and quantitate resorption by isolated osteoclasts
Authors: Walsh C.A. , Beresford J.N. , Birch M.A. , Boothroyd B. , Gallagher J.A.
Published: February 2010

A simple technique has been developed to identify the resorption lacunae excavated by avian osteoclasts in vitro. Briefly, devitalized bovine bone wafers, with cells in situ, are fixed, stained with toluidine blue, and then examined by reflected light microscopy. Resorption pits are clearly visible using a x 10 objective, even when the resorbed areas are covered by a confluent layer of cells. The technique can be used to quantify the plan area, depth, and volume of the excavations. Depth and volume are assessed using the x 50 objective lens. This technique has been used to investigate the effects of retinyl acetate and EHDP on bone resorption.
Evidence for nonrenal synthesis of 1,25-dihydroxyvitamin d in patients with inflammatory arthritis
Authors: Dr. Mawer Barbara E. , Hayes Michael E. , Still Pamela E. , Davies Michael , Lumb Geoffrey A. , Palit Jayanta , Holt Lennox P.J.
Published: February 2010

The extrarenal synthesis of 1,25-dihydroxyvitamin D [1,25-(OH)2D] is a characteristic of activated macrophages and has been demonstrated to occur in vitro in synovial fluid macrophages from patients with inflammatory arthritis. To examine whether such synthesis occurs in vivo, 19 patients with rheumatoid arthritis, 5 patient controls, and 5 healthy controls received a challenge oral dose of 250 μg 25-hydroxyvitamin D3 (25-OHD3) and the serum 1,25-(OH)2D3 response was measured. The median rise in serum 1,25-(OH)2D3 was significantly greater (22 pg/ml) in the rheumatoid patients compared to either of the control groups (8 pg/ ml), although the increase in precursor 25-OHD3 was similar in all groups. The serum 1,25-(OH)2D concentration did not rise above the normal upper limit in any of the control subjects but exceeded the normal range in 8 of the rheumatoid patients. Extrarenal 1,25-(OH)2D synthesis is substrate dependent, unlike renal 1α-hydroxylation, which is homeostatically controlled. Excessive 1,25-(OH)2D3 synthesis in the rheumatoid group on raising the 25-OHD3 concentration is indicative of nonrenal production of the hormonal metabolite. Further evidence for substrate-dependent extrarenal synthesis came from measurements of 25-OHD and 1,25-(OH)2D in paired serum and synovial fluid samples from 19 patients with inflammatory arthritis, including 15 with rheumatoid arthritis. Synovial fluid 1,25-(OH)2D was usually present at a lower concentration than serum 1,25-(OH)2D, with which it was strongly correlated (Kendall's R = 0.46, P < 0.001). Synovial fluid 1,25-(OH)2D was also correlated with synovial fluid 25-OHD (R = 0.55, P < 0.001), but serum 1,25-(OH)2D and 25-OHD were not related. At high synovial fluid 25-OHD concentrations the 1,25-(OH)2D level in synovial fluid exceeded that in serum, suggesting local synthesis of 1,25-(OH)2D within the synovium. Distribution of vitamin D metabolites between serum and synovial fluid could not be explained by the relevant concentrations of vitamin D binding protein. Together these results provide strong evidence for nonrenal synthesis of 1,25-(OH)2D in patients with inflammatory arthritis. The extent of synovial synthesis of 1,25-(OH)2D may be governed both by the severity of the inflammatory disease and by drug treatment. Such synthesis may be significant in the immunoregulatory system in the affected joint and may prove to have relevance to the development of periarticular osteoporosis in rheumatoid arthritis.
A histomorphometric study of cortical bone activity during increased weight-bearing exercise
Authors: Raab Diane M. , Crenshaw Thomas D. , Kimmel Donald B. , Smith Everett L.
Published: February 2010

To quantify cortical bone response to weight-bearing exercise, bone size, mineral content, and formation were measured at the femoral midshaft in swine. Bone formation was measured histomorphometrically on the periosteal, endosteal, and osteonal surfaces. Sedentary adult crossbred sows (3 years, 229 kg) were randomly assigned to basal (B, n = 6), control (C, n = 7), or trained (T, n = 7) groups. The basal and control groups did not exercise and were killed initially (B) or after 20 weeks (C). The trained group walked on a treadmill 20 minutes/day at 5 km/h and 5% grade, 5 days/week for 20 weeks. Bone length, area, or fat-free dry weight was not different with time (B versus C) or with training (C versus T). Periosteal modeling was stimulated by walking. Periosteal formation surface and mineral apposition rate (MAR) were greater in trained than control femora. No effects of walking were measured on the endosteal surface. Intracortical remodeling was not affected by walking. The number of labeled osteons (22.4 cm−2) was not different among groups, but osteonal MAR was greater in trained (1.18 μm/day) than control (0.96 μ/day) femora. Walking for 20 weeks in the previously sedentary sows was not a sufficient stimulus to create differences in gross measures of bone size or mineral content but did increase periosteal and intracortical MAR. The primary effect of increased exercise appeared to be osteoblast activation.
Retinoic acid modulation of mrna levels in malignant, nontransformed, and immortalized osteoblasts
Authors: Zhou Hong , Hammonds Glenn R. , Findlay David M. , Fuller Peter J. , Martin John T. , Dr. Ng Kong Wah
Published: February 2010

Clonal cell lines presumably “arrested” at a particular stage of differentiation are useful models to study the processes of differentiation in osteoblasts. UMR-201 is a presumptive preosteoblastic nontransformed rat clonal cell line with a limited life span in culture. Two immortalized cell lines, UMR-201-10A (10A) and UMR-201-10B (10B), were derived from UMR-201 by stable transfection with simian virus (SV) 40 large T antigen. This study compares the growth and profile of gene expression of the immortalized cell lines with those of UMR-201 and UMR-106-06, a rat clonal cell line with well-defined osteoblast-like phenotypic characteristics. All four cell lines constitutively expressed the mRNA for the γ, α, and β receptors for retinoic acid (RA), the growth hormone receptor, pro-α1(I) collagen, osteonectin, bone proteoglycan I, and bone morphogenetic proteins (BMP) 1 and 2A. Alkaline phosphatase mRNA was absent in the preosteoblast cell lines but was induced by treatment with 10−6 M RA, which also increased the steady-state levels of mRNA for osteopontin and BMP1. mRNA for matrix gla protein was constitutively present and further induced by RA in UMR-201 and 10B only. Messenger RNA for bone sialoprotein and bone morphogenetic protein 3 were constitutively expressed in UMR-106-06 and UMR-201 but absent in the immortalized cell lines. None of the cell lines expressed measurable mRNA for bone gla protein or bone proteoglycan II. 10B grew more rapidly than UMR-201, but unlike UMR-201, it was also able to proliferate in serum-free medium and exhibit anchorage-independent growth. In summary, this study identifies novel retinoic acid effects on gene expression in these cells. Differences noted in the expression of mRNAs between UMR-106-06 and the other cell lines may provide some insight into the sequence of expression of these phenotypic characteristics as osteoblasts differentiate.
Bradykinin-induced burst of prostaglandin formation in osteoblasts is mediated via B2 bradykinin receptors
Authors: Ljunggren Östen , Vavrek Raymond , Stewart John M. , Lerner Ulf H.
Published: February 2010

The receptor subtype mediating the bradykinin (Bk)-induced burst of prostaglandin formation in osteoblasts has been studied. Bk, but not des-Arg9-Bk, induced the formation of prostaglandin E2 and prostacyclin in neonatal mouse calvarial bones incubated for 30 minutes. Bk-induced prostaglandin synthesis in neonatal mouse calvarial bones was significantly inhibited by the B2 Bk receptor antagonist D-Arg0[Hyp3, Thi5,8, D-Phe']-Bk. The B2 Bk receptor agonists Bk and Lys-Bk, but not the B1 Bk receptor agonist des-Arg9-Bk, caused a rapid burst (5 minutes) of prostaglandin E2 and prostacyclin formation in isolated osteoblast-like cells from neonatal mouse calvarial bones and in the murine osteoblastic cell lineage MC3T3-E1. When comparing the relative potency of different kinin analogs on their stimulatory effect on prostaglandin formation in isolated osteoblast-like cells and in MC3T3-E1 cells, we found that Bk = Lys-Bk > Met-lys-Bk >>> Des-Arg9-Bk. Bk-induced prostaglandin synthesis in isolated osteoblast-like cells and in MC3T3-E1 cells was inhibited by D-Arg0[Hyp3, Thi5,8, D-Phe7]-Bk, whereas the B1 Bk receptor antagonist des-Arg9-Leu8-Bk had no effect. Total binding of 3H-Bk (3–20 nM; 4°C) to whole MC3T3-E1 cells reached a maximum after 4–5 h. An excess of nonradioactive Bk (1 μM) reduced cell-associated radioactivity by 20–30%. The B2 Bk receptor agonist Bk and the B2 Bk receptor antagonist D-Arg0[Hyp3, Thi5,8, D-Phe7]-Bk were able to reduce specific binding, but the B1 Bk receptor agonist des-Arg9-Bk was unable to reduce the specific binding of 3H-Bk to whole MC3T3-E1 cells. These findings indicate the presence of B2 Bk receptors on osteoblasts coupled to enhanced prostaglandin synthesis.
Bone mass is related to creatinine clearance in normal elderly women
Authors: Dr. Yendt Edmund R. , Cohanim Moussa , Jarzylo Stanley , Jones Glenville , Rosenberg Gilbert
Published: February 2010

We determined the relationship between bone mass and age, anthropometric variables, creatinine clearance (Ccr), and serum and urine biochemical variables in 77 normal white women (aged 41–86, mean = 67) living in their own homes. A total of 74 women were postmenopausal. Skeletal status was assessed in all subjects by x-rays of the hand with measurement of the mean combined cortical thickness (CCT) of the second metacarpal bones. In 53 women, bone mineral content of the radial shaft (RMBC) was also measured by single-photon absorptiometry (SPA) and lumbar bone mineral density (LBMD) was measured by dual-photon absorptiometry (DPA). Serum and urine biochemical variables were measured under standardized conditions on the sixth and seventh days of a controlled diet. There was a strong positive correlation between Ccr and bone mass. Although our subjects showed the expected linear decline in Ccr with age, we found that the relationship between Ccr and bone mass in the radius and lumbar spine was independent of age. On the other hand, the relationship between Ccr and CCT was not independent of age. We concluded that the relationship between Ccr and lumbar and radial bone mass is probably indicative of a relationship between glomerular filtration rate and bone mass, although this requires validation with a noncreatinine method for measurement of glomerular filtration rate. Age per se does not appear to be a cause of declining lumbar bone mass after the menopause.
Preexisting bone loss associated with ovariectomy in rats is reversed by parathyroid hormone
Authors: Liu Chung C. , Kalu Dike N. , Salerno Elena , Echon Rosanno , Hollis Bruce W. , Ray Michelle
Published: February 2010

Previous studies have demonstrated that when parathyroid hormone (PTH) administration to rats is started immediately following ovariectomy, it prevents bone loss due to ovarian hormone deficiency. In this study, we examined whether bone loss induced by ovariectomy could be reversed by pasrathyroid hormone if hormone therapy is started after the bone loss had already occurred. In the first experiment, two groups of animals were ovariectomized or sham operated, killed after 40 days, and their bones examined to ensure that bone loss occurred. In the second experiment, three groups of rats were studied. Group 1 rats were sham operated, and rats in groups 2 and 3 were ovariectomized. Each rat in group 3 received a single subcutaneous injection of 8 μg parathyroid hormone [hPTH-(1–34); Bachem, CA] per 100 g body weight per day, starting 40 days following ovariectomy. Rats in groups 1 and 2 received solvent vehicle, and all animals were sacrificed on day 60. Ovariectomized rats had lost an appreciable amount of bone 40 days after surgery, as indicated by a significant decrease in femoral and vertebral densities and calcium and an over 55% loss of cancellous bone in the tibial metaphysis. The loss of bone was reversed by intermittent PTH administration. Increased cancellous bone in the parathyroid hormone-treated ovariectomized rats was associated with increased trabecular osteoblasts, decreased trabecular osteoclasts, and increased serum osteocalcin and urinary hydroxyproline. Our findings indicate that parathyroid hormone can substantially augment bone mass after the loss due to ovarian hormone deficiency has already occurred. The hormone caused positive bone balance in vivo in ovarian hormone-deficient animals by increasing bone formation and decreasing bone resorption.
Stimulation of 1,25-dihydroxyvitamin D3 receptor gene expression in cultured cells by serum and growth factors
Authors: Krishnan Aruna V. , Feldman David
Published: February 2010

The abundance of 1,25-dihydroxyvitamin D3 receptors (VDR) in bone cells has been shown to vary in direct relation to the rate of cell proliferation. In this study we further explored this upregulation of VDR as it relates to the mitogenic response using NIH-3T3 mouse fibroblasts and MCF-7 human breast cancer cells as model systems. Serum and growth factors, such as EGF, high concentrations of insulin (2 μM), and IGF-I, were mitogenic and stimulated the proliferation of both cells types. These factors also caused significant increases in VDR levels as measured by ligand binding assays, which preceded the rise in cell proliferation rate measured by [3H]thymidine incorporation. Serum and growth factors increased the abundance of VDR but did not affect the concentrations of other steroid receptors in MCF-7 cells. Mouse cells have been reported to have several VDR mRNA transcripts. Our northern blot analysis revealed three mRNA species at approximately 7.5, 4.4, and 3 kb of which the 4.4 kb species was the most prominent and the 7.5 kb the least. Serum and growth factor stimulation of quiescent 3T3 cells led to significant increases in all the transcripts, suggesting that the upregulation occurs at the level of VDR mRNA expression. A time course analysis of serum stimulation in 3T3 cells showed that the mRNA species reached peak levels 4 h after serum addition. When serum stimulation was carried out in the presence of the protein synthesis inhibitor cycloheximide, the 3 kb transcript as well as the 7.5 kb transcript were superinduced but the stimulation of the 4.4 kb transcript was inhibited. Hydroxyurea (an inhibitor of DNA synthesis) and colchicine (a microtubule disruptive agent) had no effect on serum stimulation of the mRNA species, indicating that the enhanced expression of the VDR gene is not dependent upon the S, G2, or M phase of the cell cycle. The results suggest that the VDR gene appears to be one of a set of early genes activated when quiescent cells are stimulated by a mitogenic signal.
Voltage-dependent phosphate transport in osteoblast-like cells
Authors: Luong KHANH V.Q. , Green Jacob , Kleeman Charles R. , Yamaguchi Dean T.
Published: February 2010

Phosphate ion (Pi) in sufficient concentrations is crucial for bone mineralization. The osteoblast (OB) may be responsible for the transport of Pi into the bone interstitium, where mineralization occurs. We previously characterized a Na+-dependent Pi transporter (NaPi) in the osteoblastic UMR-106–01 cell line. In the present study, the alteration of Na+-dependent Pi transport by changes in membrane potential was investigated. Depolarizing the cells with increasing concentrations of ambient K+ and valinomycin resulted in a progressive decline in Na+-dependent Pi uptake to a maximum of 28% at a membrane potential of −18 mV compared to control Na+-dependent Pi uptake at a membrane potential of approximately −60 mV. Hyperpolarizing the cells with SCN− increased Na+-dependent Pi uptake over control by 50% at an SCN− concentration of 70 mM. Determination of membrane potential by using the fluorescent probe, DiSC3(5), showed that the addition of Pi to cells in Na+-containing medium resulted in a small depolarization. These data show that NaPi activity can be altered by membrane potential changes and that the initiation of Na+-dependent Pi uptake is associated with depolarization of the plasma membrane of UMR-106–01 cells. Taken together, the cotransport of Na+ and Pi results in the movement of a net positive charge into the cell.
Effects of 17β-estradiol on calcitonin secretion and content in a human medullary thyroid carcinoma cell line
Authors: Lazaretti-Castro Marise , Grauer Andreas , Mekonnen Yalem , Raue Friedhelm , Ziegler Reinhard
Published: February 2010

The presence of a direct estrogen effect on calcitonin secretion is controversial. Because most of the data available were obtained from complex in vivo systems, we chose an in vitro approach to assess the problem. Using a human C cell carcinoma cell line (TT cells) with well-documented estrogen receptors, we investigated the effect of 17β-estradiol (E2) on basal and stimulated calcitonin secretion, on calcitonin content, and on total cellular protein. After short (30 and 180 minutes) and long-term (24 h to 6 days) incubation of the cells with different concentrations of E2 (from 0.01 to 100 nM) we observed no stimulatory but a transient dose-dependent inhibitory effect on CT secretion and content. The nadir of the effect on CT secretion appeared at 24 h, demonstrating a reduction to 80.5 ± 7.8% of control at 1 nM and to 59.1 ± 15% of control at 100 nM E2. After 72 h, the CT levels of the E2-exposed groups returned to control levels. The acute stimulation of the cells with TPA plus forskolin after preincubation with E2 up to 6 days showed no difference in the increment of CT release compared to the control groups. Additionally, E2 had a dose-dependent stimulatory effect on cell protein content. The data demonstrate the absence of a direct stimulatory effect of E2 on CT secretion, revealing a dose-dependent inhibitory effect on CT secretion and content.
Lack of effects of neutralization of parathyroid hormone-related protein on calcium homeostasis in neonatal mice
Authors: Kukreja Subhash C. , D'Anza James J. , Melton Mary E. , Wimbiscus Sarah A. , Grill Vivian , Martin John T.
Published: February 2010

Large quantities of parathyroid hormone-related protein (PTHrP) are present in the milk of various species. It has been suggested that PTHrP may play a role in neonatal calcium homeostasis. In the present study we evaluated the effect of neutralization of amino-terminal PTHrP activity by passive immunization in 1-day-old mouse pups. Neutralization of amino-terminal PTHrP activity had no significant effect on serum calcium or whole-body calcium content in the neonatal mice. In additional studies, we demonstrated that subcutaneous administration of PTHrP-(1–34) increased serum calcium, whereas oral administration had no significant effect in 3-day-old pups. The studies therefore demonstrate that the amino terminus of PTHrP may not play a significant role in neonatal calcium homeostasis. Local effects of PTHrP cannot be excluded by the results of the present study.
Regulation of osteoblast proliferation by leukemia inhibitory factor
Authors: Lowe Carolyn , Cornish Jill , Callon Karon , Martin John T. , Dr. Reid Ian R.
Published: February 2010

We recently showed that leukemia inhibitory factor (LIF) stimulates 45Ca release from neonatal mouse calvariae in vitro and that it increases DNA and protein synthesis in this model. To elucidate further the actions of LIF on bone we now report the effects of this cytokine on DNA synthesis and cell proliferation in isolated fetal rat osteoblasts and in the osteogenic sarcoma cell line, UMR-106. In both actively growing and growth-arrested rat osteoblasts, LIF stimulated [3H]thymidine incorporation in a dose-dependent manner. The increase in DNA synthesis was time dependent, was associated with an increase in the number of osteoblasts, and was not blocked by indomethacin. LIF-treated cells showed reduced [3H]thymidine incorporation in comparison with control, as they approached confluence, possibly because of the increased cell density in the LIF-treated cultures.

In UMR-106 cells, treatment with LIF inhibited [3H]thymidine incorporation in both actively growing and growth-arrested cultures. The effect was dose dependent and sustained with time. There was a corresponding decrease in cell numbers. It is concluded that although LIF causes an early stimulation of proliferation in isolated osteoblasts, it has opposing effects on UMR-106 cells. It is not possible to determine which of these effects is more relevant to the actions of LIF in vivo. The demonstration of a LIF effect on both these cell types, however, provides further evidence that this cytokine acts directly on osteoblasts.