bluuebunny/crossref_metadata_2025 huggingface dataset:
High-conductance anion channels in embryonic chick osteogenic cells
Authors: Ravesloot Jan H. , van Houten Ron J. , Ypey Dirk L. , Nijweide Peter J.
Published: February 2010

Patch-clamp measurements done on excised membrane patches obtained from 1-5 day cultured embryonic chick osteoblasts, osteocytes, and periosteal fibroblasts revealed the existence of a high-conductance anion channel: 371 ± 63 pS when measured under symmetrical 158 mM CI− conditions. The channel frequently displayed subconductance levels. The ion selectivity of the channel expressed as the (an)ion to chloride permeability ratio was as follows: CI− (1.0) > methylsulfate− (0.71) > gluconate− (0.25) > glutamate− (0.17) > Na+ = K+ (0.10). In addition, the channel had a significant permeability for inorganic phosphate ions. The channel was found in about 1% of the cell-attached patches, which indicates that the channel is under the control of as yet unknown intracellular factors. Once activated by patch excision, the channel was voltage dependent and active at potentials close to 0 mV. At potentials outside the range of ± 10 mV channel activity decreased. This process proceeded faster at increasing membrane potentials of either polarity. Returning to potentials close to 0 mV caused reopening of the channels within seconds if the preceding voltage step led to complete closure of the channels. Channel activity did not depend noticeably on intracellular and extracellular Ca2+ ions. The channel is not unique to (chick) osteogenic cells but has been demonstrated in excised patches obtained from excitable and other nonexcitable cells. Although its presence in a wide variety of cell types suggests that the channel plays a general role in as yet unknown cell physiologic processes, the channel may also have specific functions in osteogenic cells, for example providing a pathway for phosphate ions during mineralization.
Characterization of a tartrate-resistant acid phosphatase (ATPase) from rat bone: Hydrodynamic properties and N-terminal amino acid sequence
Authors: Ek-Rylander Barbro , Bergman Tomas , Andersson Göran
Published: February 2010

Certain physicochemical properties of rat bone tartrate-resistant acid ATPase (TrATPase), including the size and shape of the enzyme, potential subunit composition, and detergent binding, have been elucidated. SDS-polyacrylamide gel electrophoresis in combination with immunoblot analysis showed that the bone TrATPase has a molecular weight of 33,000 D and is composed of disulfide-linked polypeptides of 20,000 and 16,000 D. The enzyme contains 1.7 mol Fe per mol enzyme. Hydrodynamic studies allowed calculation of the Stokes radius (24 Å), the sedimentation coefficient (3.19S), the partial specific volume (0.748 ml/g), the frictional ratio (0.995), and the axial ratio (1.0). The amount of detergent bound to the protein was determined to 4 mol of Triton X-100 per mol enzyme. The molecular weight of bone TrATPase derived from these parameters was 31,900 D. N-terminal amino acid sequence analysis of the Mr 20,000 subunit indicated a high degree of similarity with TRAP enzymes from spleen, uterus, placenta, hairy cell leukemia, and osteoclastoma.

It is concluded that rat bone TrATPase belongs to the type 5 (tartrate-resistant and purple) acid phosphatase family. The similarities in the N-terminal amino acid sequences, iron content, and physicochemical properties of TRAP enzymes indicate a close structural relationship between type 5 acid phosphatases expressed in different tissues. The findings that TrATPase has a spherical shape and binds low amounts of detergent suggest that the enzyme is a soluble protein, compatible with the view that TrATPase is secreted by the osteoclast.
A systemic acceleratory phenomenon (SAP) accompanies the regional acceleratory phenomenon (RAP) during healing of a bone defect in the rat
Authors: Mueller Martina , Schilling Tobias , Minne Helmut W. , Ziegler Reinhard
Published: February 2010

The rate of remodeling in the region of a bone defect exceeds normal tissue activity. It was Frost who described this reaction as a regional acceleratory phenomenon (RAP). We investigated the local healing process with rats with a burr hole defect (1.2 mm in diameter) in the left tibia. We differentiated an initial phase of bone formation followed by a phase of predominant resorption. To determine whether this regional enhancement of bone formation would result in a systemic impact on bone metabolism, we analyzed both tibiae and femora and the fourth lumbar vertebra. On day 7 both femora of rats with the tibial defect showed a significant increase in computerized x-ray density, dry weight, ash weight, and Ca2+ content. Both tibiae and the fourth lumbar vertebra showed a significant increase in mineralizing surface, mineral apposition rate, and bone formation rate. Because of these results we conclude that a systemic acceleratory phenomenon (SAP) accompanies the RAP.

SAP affects only the cancellous, but not the cortical bone compartment. SAP is associated closely with the occurrence of woven bone during the formation phase of the healing process. Thus we assume that woven bone formation plays a pivotal role in the mediation of SAP.
Measurement of mechanical properties of bone material in vitro by ultrasound reflection: Methodology and comparison with ultrasound transmission
Authors: Antich Peter P. , Anderson Jon A. , Ashman Richard B. , Dowdey James E. , Gonzales Jerome , Murry Robert C. , Zerwekh Joseph E. , Pak Charles Y. C.
Published: February 2010

An ultrasound reflection technique was designed and implemented to study the mechanical properties of bone material. The technique uses the fact that an ultrasound beam produced in water undergoes total internal reflection off a bone sample at a critical angle formally related to the velocity of a pressure wave in bone. When the plane of scattering is rotated around the normal to the sample surface, the critical angle varies with a periodic dependence dictated by the intrinsic symmetry of the bone structure at the point being examined.

Most current measurements of sound velocity are made using transmission techniques. A double-blind intercomparison between this technique and a transmission technique, which was previously validated against tensile mechanical testing, was performed for samples of isotropic materials and of human cortical bone. Strong correlations were found for both sets of samples. For the isotropic materials the velocities were approximately equal, but for bone they were on average 11% higher in reflection than in transmission. This was the result both of the higher frequency employed in reflection (3.5 rather than 2.25 MHz) and of the different effects of sample imperfections on the two measurements. In particular, the reflection technique used in this work studied the surface of the sample, but the ultrasound beam in the transmission method propagated through its interior.

In assessing the mechanical properties of bone specimens by ultrasound, the reflection technique samples a discrete bone surface element and the transmission method analyzes the entire volume of the specimen. Thus the reflection technique may yield a measure of the mechanical property of bone trabeculae that is largely unaffected by the mass of the entire specimen, but mass and the structural density of the specimen affect the transmission method.
The impact of menopausal age on future fragility fracture risk
Authors: Gärdsell Per , Johnell Olof , Nilsson Bo E.
Published: February 2010

Altogether 733 postmenopausal women were interviewed with regard to their age at menopause. Subsequent fragility fractures over an 11 year period were recorded. Fragility fractures had occurred in 212 women. These women had an earlier menopause, but the deviation from the nonfracture group was significant only in those who were <70 years at the beginning of the fracture catchment period. Women 50–69 years of age, in the lowest quartile of menopausal age, sustained 50% more fragility fractures than those in the highest quartile. In older women menopausal age does not predict fragility fractures. Women <70 with an early menopause had a significantly lower initial bone mass.
The role of DNA synthesis in the responses of fetal rat calvariae to cortisol
Authors: Lukert Barbara , Mador Anne , Raisz Lawrence G. , Kream Barbara E.
Published: February 2010

To determine the extent to which the effects of cortisol on collagen synthesis in 21 day fetal rat calvariae are linked to its effects on cell replication, calvariae were cultured for 24–72 h with 0.1 and 1 μM cortisol in the presence or absence of 1 mM hydroxyurea (HU) or 30 μM aphidicolin (APC), inhibitors of DNA synthesis. The incorporation of [3H]proline into collagenase-digestible protein (CDP) and [3H]thymidine into DNA were measured during the last 2 h of culture. At 24 h HU and APC decreased thymidine incorporation by >90%, and this remained low for the duration of culture. In contrast, cortisol reduced thymidine incorporation by only 44% at 72 h. Although cortisol caused a 24 h stimulatory effect and a 48 and 72 h inhibitory effect on CDP labeling and the percentage of collagen being synthesized (PCS), HU, and APC had no effect on basal CDP labeling or PCS over the 72 h culture period. Cortisol caused parallel alterations in the steady-state levels of α-1(I) procollagen mRNA, suggesting that its effects occur at the pretranslational level. At 24 h HU and APC did not prevent the stimulatory effect of cortisol on CDP labeling and PCS. At 48 h the inhibitory effects of cortisol on CDP labeling and PCS were observed in the presence of APC but not in the presence of HU. At 72 h the inhibitory effects of cortisol on CDP labeling and PCS were still observed in the presence of HU and APC. The inhibitory effect of cortisol on CDP labeling was only partially reversed 48 h after removing cortisol from calvariae treated with hormone for 48 h. These data show that DNA synthesis inhibitors by themselves do not mimic the effects of cortisol on collagen synthesis nor do they prevent the early stimulation and the late inhibition of collagen synthesis by cortisol. Thus we conclude that the long-term inhibitory effect of cortisol on collagen synthesis in calvariae is largely independent of cell replication. The component of collagen synthesis that is reversible may represent the direct inhibitory effect of cortisol on differentiated osteoblasts. The nonreversible component of inhibition may be due to a more permanent effect of cortisol on cell populations within the calvariae. One possibility is that cortisol inhibits differentiation of osteoprogenitor cells to the osteoblast lineage leading to decreased osteoblast number and a subsequent inhibition of collagen production.
Serum ionized calcium, as well as phosphorus and parathyroid hormone, is associated with the plasma 1,25-dihydroxyvitamin D3 concentration in normal postmenopausal women
Authors: Dawson-Hughes Bess , Harris Susan , Dallal Gerard E.
Published: February 2010

Serum parathyroid hormone (PTH) and low-normal serum phosphorus (P) concentrations have well-known trophic effects on renal 1-hydroxylase. A role for serum ionized calcium (Ca2+) in the day-to-day regulation of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has not been identified in normal humans. The associations between serum Ca2+, PTH, P, and plasma 1,25-(OH)2D3 were evaluated in a cross-sectional study of 275 healthy postmenopausal women. Partial correlations of Ca2+, PTH, and P (each controlled for the other two) with 1,25-(OH)2D3 were sought within quintiles of Ca2+ At low-normal concentrations (<1.26 mmol/liter, quintile 1) Ca2+ was inversely correlated with 1,25-(OH)2D3 (rp = -0.30, p = 0.028). At midnormal concentrations Ca2+ was not significantly correlated with 1,25-(OH)2D3. At high-normal concentrations >1.32 mmol/liter, quintile 5) Ca2+ attenuated the positive associations of both PTH and low-normal P with 1,25-(OH)2D3. In quintile 5 Ca2+, PTH, and P together accounted for none of the variability in 1,25-(OH)2D3 (R2 = 0.03, p = 0.671). Women with Ca2+ below 1.32 mmol/liter were next examined by quintile of P. As expected, at low-normal concentrations (<1.03 mmol/liter, quintile 1) P was significantly correlated with 1,25-(OH)2D3 (rp = -0.32, p = 0.047). The association between PTH and 1,25-(OH)2D3 was statistically significant only at midnormal concentrations of P (rp = 0.52, p = 0.001, quintile 3). We conclude that Ca2+, along with PTH and P, is associated with the plasma concentration of 1,25-(OH)2D3 in normal postmenopausal women.
Fecal calcium density: A measure of calcium compliance
Authors: Heaney Robert P.
Published: February 2010

We evaluated fecal calcium density (mass of calcium per g dry weight of feces) as a measure of compliance with a prescribed calcium intake regimen using 4 day fecal pools collected on a metabolic research unit from subjects ingesting measured, constant intakes. Fecal calcium density was highly correlated with intake (r = 0.897, P < 0.001). Intake estimates based on fecal calcium density exhibited a standard error of the mean equal to 3.76 mmol calcium. Since a typical calcium supplement table contains 12.5 mmol calcium, the measurement of fecal calcium density is sensitive enough to detect regular omission of one or more pills daily. Applicability of this approach to convenience samples of feces was evaluated in 15 individuals by testing homogeneity of fecal calcium density values on up to six different 3–9 g portions (wet weight) of each volunteer's fecal sample. The within-sample coefficient of variation was 9.5% for all subsamples and 7.3% for samples from individuals with intakes above 25 mmol calcium per day. Thus feces are reasonably homogeneous in regard to calcium density. Accordingly, reasonably small fecal collections should suffice for its measurement.
Procalcitonin NH2-terminal cleavage peptide has no mitogenic effect on normal human osteoblast-like cells
Authors: Hassager Christian , Bonde Susan K. , Anderson Marlys A. , Rink H. , Spelsberg Thomas C. , Riggs B. Lawrence
Published: February 2010

The NH2-terminal cleavage peptide of procalcitonin (N-proCT) recently was reported to be a bone cell mitogen (Burns DM et al., Proc Natl Acad Sci USA 86:9519–9523, 1989). We have investigated the effect of N-proCT on the proliferation of normal human cells that have the phenotype of mature osteoblasts (hOB cells). N-proCT treatment for 24, 48, or 96 h in concentrations from 1 nM to 1 μM did not significantly increase [3H]thymidine uptake (means ranged from -19% to 38% of control, no significant differences) in hOB cells (6–10 cell strains per experiment) plated at four different densities. However, the hOB cells responded significantly to treatment with transforming growth factor β (3 ng/ml), bovine insulin (300 μg/ml), or 30% fetal calf serum, which were included in all experiments as positive controls. The [3H]thymidine uptake data were confirmed in a direct cell count experiment tested at 96 h. Thus our data do not support the hypothesis that N-proCT is a potent mitogen for normal human osteoblasts.
1,25-Dihydroxyvitamin D reduces parathyroid hormone receptor number in ROS 17/2.8 cells and prevents the glucocorticoid-induced increase in these receptors: Relationship to adenylate cyclase activation
Authors: Dr. Titus Louisa , Jackson Elizabeth , Nanes Mark S. , Rubin Janet E. , Catherwood Bayard D.
Published: February 2010

We have previously shown that 1,25-dihydroxyvitamin D [1,25-(OH)2D3] and glucocorticoid modulate adenylate cyclase activation by PTH in osteoblast-like cells. Here we examine whether steroid effects on PTH receptor density explain the modulation of PTH action. Receptor assays were performed on late logarithmicphase monolayers of ROS 17/2.8 cells using human PTH-like peptide (hPLP) as radioligand. Kd and receptor density were computed from competition of tracer amounts of [125I-Tyr36]hPLP-(1–36) with unlabeled hPLP-(1–36) (0.1–30 nM). Steroid treatment had little or no effect on affinity for ligand. Pretreating cells with 10 nM 1,25-(OH)2D3 for 48 h decreased PTH receptor number to 17% of control values. Treating cells with 10 nM of the glucocorticoid triamcinolone acetonide (TRM) increased receptor number 10-fold, but simultaneous treatment with 1,25-(OH)2D3 (10 nM) completely prevented this receptor increase. Steroid effects required 13–18 h of treatment. Dose-response relationships for steroid modulation, determined from binding at 0.17 nM radioligand, indicated an EC50 of 0.3 nM for glucocorticoid augmentation of PTH receptor number and 0.02 nM for 1,25-(OH)2D3 reduction of receptor number in the presence or absence of the maximum TRM effect. The initial rate of cAMP production by receptor-saturating concentrations of PTH was 11,500 molecules per receptor per minute in untreated cells, comparable to reported turnover numbers for mammalian adenylate cyclase. Control experiments were validated measuring cAMP in intact cells as an indicator of adenylate cyclase activity. Cyclic AMP production was reduced 63% by 1,25-(OH)2D3 (10 nM) treatment. Glucocorticoid (10 nM) enhanced cAMP production twofold but reduced cAMP generation per receptor by 80%. We conclude that 1,25-(OH)2D3 reduction of PTH receptor number modulates ROS cell responses to PTH and prevents glucocorticoid-enhanced PTH action. The data also suggest that other components of the adenylate cyclase complex limit amplification of the PTH signal when PTH receptor number is elevated by glucocorticoid treatment.
Inhibition of 1,25-(OH)2D3- and 24,25-(OH)2D3-dependent stimulation of alkaline phosphatase activity by A23187 suggests a role for calcium in the mechanism of vitamin D regulation of chondrocyte cultures
Authors: Schwartz Z. , Langston G.G. , Swain L.D. , Boyan Barbara D.
Published: February 2010

This study used the ionophore, A23187, to examine the hypothesis that the regulation of alkaline phosphatase and phospholipase A2 activity by vitamin D3 metabolites in cartilage cells is mediated by changes in calcium influx. Confluent, fourth-passage cultures of growth zone and resting zone chondrocytes from the costochondral cartilage of 125 g rats were incubated with 0.01-10 μM A23187. Specific activities of alkaline phosphatase and phospholipase A2 were measured in the cell layer and in isolated plasma membranes and matrix vesicles. There was an inhibition of alkaline phosphatase specific activity at 0.1 μM A23187 in resting zone cells and at 0.1 and 1 μM in growth zone chondrocytes. At these concentrations of ionophore, the 45Ca content of the chondrocytes was shown to increase. Both the plasma membrane and matrix vesicle enzyme activities were inhibited. There was no effect of ionophore on matrix vesicle or plasma membrane phospholipase A2 in either cell type. In contrast, alkaline phosphatase activity is stimulated when growth zone chondrocytes are incubated with 1,25-(OH)2D3 and in resting zone cells incubated with 24,25-(OH)2D3. Phospholipase A2 activity is differentially affected depending on the metabolite used and the cell examined. Addition of ionophore to cultures preincubated with 1,25-(OH)2D3 or 24,25-(OH)2D3 blocked the stimulation of alkaline phosphatase by the vitamin D3 metabolites in a dose-dependent manner. The effects of ionophore were not due to a direct effect on the membrane enzymes since enzyme activity in isolated membranes incubated with A23187 in vitro was unaffected. These results suggest a role for calcium in the action of vitamin D metabolites on chondrocyte membrane enzyme activity but indicate that mechanisms other than merely Ca2+ influx per se are involved.
Depression of osteoblastic activity in immobilized limbs of suckling rats
Authors: Weinreb Miron , Rodan Gideon A. , Thompson David D.
Published: February 2010

Recently we characterized the immobilization-related osteopenia in adult rats and showed that it is caused by increased bone resorption and decreased bone formation (Weinreb et al. 1989 Bone 10:187). To assess the effect of age on disuse osteopenia, this study investigated the effects of immobilization on bone turnover in very young, suckling rats. The 15-day-old rats underwent unilateral hind limb immobilization by sciatic neurectomy; the contralateral limb was left intact and served as control. Experimental or sham-operated animals were killed after 0, 2, 4, or 12 days postsurgery. Dry, fat-free weight and ash weight were determined in both femora, and both tibiae were subjected to static and dynamic histomorphometry. Immobilization caused a progressive deficit in bone mass in the immobilized limb compared to the contralateral intact limb but did not affect femoral longitudinal growth. The total mineral content in the immobilized femora was 13.6% less than that in the intact limb by day 12. Concomitantly, tibial cancellous bone area and perimeter declined in the immobilized limb by 37.3 and 32.2%, respectively. This reduction in bone mass in the tibiae of immobilized limbs was associated with increased bone resorption, expressed as osteoclast perimeter, number of osteoclasts per mm surface, and number of osteoclasts per mm2 tissue area. Bone formation was reduced as a result of impaired osteoblast activity as evidenced by (1) decreased endocortical and trabecular mineral apposition rate; (2) reduced trabecular mineral formation rate; (3) decreased percentage of ash of the femoral dry weight; and (4) increased volume of unmineralized osteoid in the tibial metaphysis. These data indicate that osteoblasts in very young rats are sensitive to mechanical disuse and exhibit impaired function within a short period after the onset of immobilization. They also suggest that mechanical unloading inhibits mineralization to a greater extent than matrix deposition, leading to the production of hypomineralized bone.
Effects of systemic aluminum on the resolution of a uremic and dietary phosphorus-dependent model of uremic osteomalacia in rats
Authors: Lieuallen Warren G. , Dr. Weisbrode Steven E.
Published: February 2010

We have developed a model of osteomalacia that is dependent on both uremia and the feeding of a diet low in phosphorus and that can be reversed by subsequent dietary phosphorus repletion. The objectives for this study were to use this model to investigate the role of aluminum (Al) in both the induction and resolution of osteomalacia. Adult male Sprague-Dawley rats were five-sixths nephrectomized and fed either low or normal dietary phosphorus, both with and without intraperitoneal Al injections. Uremic rats fed low phosphorus developed osteomalacia characterized by increased osteoid surface, volume, and thickness and osteoid maturation time and decreased mineralizing surface. Al-treated uremic rats fed low phosphorus were similarly affected, developing increased osteoid volume and thickness and osteoid maturation time and decreased osteoblastic surface, mineralizing surface, and bone formation rate. In addition, they had a significantly increased Al-positive surface. Al-treated uremic rats fed normal phosphorus had only increased osteoid thickness and aluminum-positive surface and decreased osteoblastic surface. Osteomalacic rats continuously treated with Al during the induction and phosphorus repletion stages had increased growth plate thickness, osteoid volume and thickness, and Al-positive surface and decreased osteoblastic and mineralizing surface. Mineralization in these rats was impaired to such a degree that no detectable double labels were present. Osteomalacic rats treated with Al during the induction phase but not during phosphorus repletion had increased osteoid surface and volume and Al-positive surface and decreased osteoblastic and mineralizing surface. Double labels were not detectable in these rats, either. These results indicate that the osteomalacia induced in uremic rats fed low phosphorus was similar in rats with and without Al treatment. However, Al prevented the reversal of this osteomalacia by phosphorus repletion, even when Al administration was discontinued. The impairment of mineralization by Al in this model of osteomalacia suggests that Al-positive bone surfaces are not an epiphenomenon in osteomalacia associated with uremia.
Solid-phase synthesis and biologic activity of human parathyroid hormone(1–84)
Authors: Goud Nagana A. , McKee Roberta L. , Dehaven Patrica A. , Huelar Elizabeth , Syed Mustaqim M. , Goud Rajeswari A. , Gibbons Susan W. , Fisher John E. , Levy Jay J. , Rodkey John A. , Bennett Carl , Ramjit Harri G. , Caporale Lynn H. , Caulfield Michael P. , Dr. Rosenblatt Michael
Published: February 2010

We have chemically synthesized the full-length, 84 amino acid, human parathyroid hormone (hPTH) on a > 100 mg scale by the Merrifield solid-phase technique of stepwise peptide synthesis using a benzhydrylamine support. The peptide was purified by high-performance liquid chromatography and found to be > 96% pure. The authenticity of the sequence of the synthetic peptide was confirmed by repetitive Edman degradation. Furthermore, tryptic digestion of hPTH generated the predicted fragments. The synthetic full-length hormone was evaluated for biologic activity in assays of PTH receptor binding and stimulation of adenylate cyclase activity (using bovine renal cortical membranes and rat and human bone cells). Synthetic hPTH(1–84) was found to be highly potent in binding to PTH receptors (Kb = 1–25 nM) and stimulating adenylate cyclase (Km = 1–14 nM). The availability of significant quantities of synthetic full-length hPTH and future analogs will permit widespread use in multiple in vitro and in vivo assays to delineate their spectrum of biologic properties. Available supplies of the synthetic hormone will also enable evaluation of the effectiveness of PTH antagonists at inhibiting the action of native sequence hormone at its receptors.
Evidence for interleukin-1β production by cultured normal human osteoblast-like cells
Authors: Keeting Philip E. , Rifas Leonard , Harris Steven A. , Colvard Douglas S. , Spelsberg Thomas C. , Peck William A. , Dr. Riggs Lawrence B.
Published: February 2010

To determine if bone cells produce interleukin-1β (IL-1β), a potent bone resorption-stimulating agent, we studied well-characterized, nearly homogeneous cultures of normal human osteoblast-like (hOB) cells. With four strains of such cells, vehicle-treated cultures produced minimal IL-1β (mean ± SEM, 1.3 ± 0.3 pg/ml per 106 cells per 24 h) and showed dose-dependent (r = 0.99) increases to 2.2 ± 0.7, 5.0 ± 0.9, or 17.8 ± 6.7 pg/ml, respectively, after treatment with lipopolysaccharide (LPS) at 3, 10, or 30 μg/ml (for increases after 10 and 30 μg/ml treatments, P < 0.05). After treatment with tumor necrosis factor α (TNF-α at 10 U/ ml, IL-1β increased to 16.2 * 3.7 pg/ml (P < 0.05). Neither 17β-estradiol nor bovine parathyroid hormone(1–34) (each at 10 nM), alone or in combination with LPS or TNF-α, affected IL-1β release. Northern blot analysis of total cellular RNA preparation revealed a single hybridization band at 1.9 kb when probed with a partially deleted cDNA for human IL-1β. The steady-state IL-1β mRNA levels showed a significant increase with LPS treatment and a lesser increase with TNF-α treatment in hOB cells. Moreover, TNF-α produced an even greater increase in IL-1 mRNA in HOBIT cells, a well-differentiated clonal cell line derived from normal hOB cells transfected with the SV40 large T antigen. We conclude that human cells of the osteoblast lineage produce IL-Iβ in response to well-recognized stimuli for IL-1 release from responsive tissue. Therefore, IL-1 may play an important role in the local regulation of bone remodeling.
Uptake of fluorine in cortical and trabecular bone
Authors: McNeill K.G. , Coote G.E. , Hitchman A.J.W.
Published: February 2010

Nuclear magnetic resonance techniques can measure the fluoride levels in bone of the finger after a patient has ingested F in the treatment of osteoporosis, but does uptake in cortical bone reflect uptake in the critical trabecular bone? Investigation has been made of the relative uptake of fluoride from drinking water into trabecular and cortical bone of the rat. For fine detail of the uptake of F into the femur and vertebra, microprobe techniques were used with a spatial resolution of 10 μm; for broader studies, treating the femur as representative of cortical bone and the vertebra as typical of trabecular bone, chemical techniques using spectroscopy and ion-selective electrodes were employed. The conclusion is that in the rat uptake of F by cortical bone is indicative of uptake by trabecular bone, and that therefore as a working hypothesis NMR measurement of F in the finger may be taken as reflecting uptake of F by trabecular bone.
A new approach to defining normal vertebral dimensions
Authors: Black Dennis M. , Cummings Steven R. , Stone Katie , Hudes Estie , Palermo Lisa , Steiger Peter
Published: February 2010

We developed a method for estimating the mean and standard deviation of ratios of normal vertebral heights from a sample that includes people with and without vertebral fractures. This method assumes that the measurements in normal vertebrae have a Gaussian distribution and that, for any vertebral level, the prevalence of abnormal measurements is less than 10%. Under these assumptions, normal values for nonfractured vertebrae can be estimated from several statistical properties of Gaussian distributions. We applied these methods to the lateral spinal radiographs of 2992 women aged 65–70 years who were recruited from population-based listings. The estimated means and standard deviations for ratios of dimensions in nonfractured vertebrae were very similar to those based on studies of premenopausal women. Our method may be useful for defining normal values from large populations that include normal and abnormal women, does not require x-rays of normal premenopausal women, avoids the potential biases of defining normality based on qualitative judgment, and can be applied to other types of physical and biochemical measurements.
Nonhypercalcemic 1,25-(OH)2D3 analogs potently induce the human osteocalcin gene promoter stably transfected into rat osteosarcoma cells (ROSCO-2)
Authors: Morrison Nigel A. , Eisman John A.
Published: February 2010

1,25-Dihydroxyvitamin D3[1,25-(OH)2D3] is the active hormonal form of vitamin D3 and has potent effects on bone and calcium regulation. Over the past decade it has become apparent that 1,25-(OH)2D3 has other effects on cellular proliferation that potentially could be developed for therapy in human malignancy. Since the hypercalcemic effects of 1,25-(OH)2D3 have limited that use in the human, novel nonhypercalcemic analogs of 1,25-(OH)2D3 have been synthesized. The molecular mechanism of this divergence in these antiproliferative and calcium-regulating actions is unexplained. We have previously examined the human bone-specific gene osteocalcin as a model of the molecular mechanisms of vitamin D action in bone and have shown that induction of the osteocalcin gene by 1,25-(OH)2D3 is mediated through an unique and complex palindromic region of the promoter similar to but distinct from those of other steroid hormone-responsive elements. Using an osteosarcoma cell line permanently transfected with the vitamin D-responsive promoter of the human osteocalcin gene linked to a “reporter” gene, we have shown that there is a dose-dependent induction of CAT activity by 1,25-(OH)2D3 and that the potencies of vitamin D metabolites and analogs are comparable to those found in other vitamin D bioassays. Furthermore, vitamin D analogs, including MC-903, 22-oxa-1,25-(OH)2D3, and Δ22–1,25S,26-trihydroxyvitamin D3, which effect cellular differentiation but lack hypercalcemic activity in vivo, exhibit osteocalcin promoter inductive actions virtually identical to those of 1,25-(OH)2D3. Consideration of these and other data support the hypothesis that the divergent effects of such analogs on differentiation and calcium homeostasis reflect pharmacokinetic differences in vivo rather than distinct 1,25-(OH)2D3-sensitive pathways.
Adenylate cyclase stimulating activity immunologically similar to parathyroid hormone-related peptide can be extracted from fetal rat long bones
Authors: Wolf Nicole Nijs-de , Pepersack Thierry , Corvilain Jacques , Karmali Rafik , Bergmann Pierre
Published: February 2010

We observed that culture medium conditioned with fetal rat long bones contained peptides immunologically related to the parathyroid hormone-related peptide of malignancy (PTHrP) and stimulated cyclic AMP production in canine renal cortical membranes. Because the adenylate cyclase stimulating activity (CSA) of the medium increased when bone resorption was stimulated, it was suspected that these peptides were stored in the matrix and released during the resorption process. In this work, we extracted the noncollagenous proteins of fetal rat long bones and found that the extract contained significant amounts of CSA. The biologic activity of the extract was abolished after trypsin digestion and eluted at 24 and 37 kD on filtration HPLC. The CSA of bone extract and of both HPLC peaks could be inhibited by 3–34 and 7–34 parathyroid hormone analogs. It was not blocked by an antiserum directed against the N-terminal region of parathyroid hormone, but it was significantly inhibited after an overnight preincubation with an antiserum directed against the 1–11 fragment of PTHrP. One band migrating at 18 kD could be visualized after SDS-PAGE electrophoresis of bone extract and immunoblotting with the anti-PTHrP antiserum. We conclude that an adenylate cyclase stimulator immunologically similar to PTHrP is present in the matrix of fetal rat long bones. Adenylate cyclase stimulating peptides of lower molecular weight found in bone-conditioned medium could be active fragments formed by proteolysis during the resorption process.
Iliac trabecular bone formation predicts radial trabecular bone density changes in type 1 osteoporosis
Authors: Hesp Richard , Arlot Monique E. , Edouard Claude , Bradbeer Jeremy N. , Meunier Pierre J. , Dr. Reeve Jonathan
Published: February 2010

In 28 patients with idiopathic or postmenopausal type 1 (spinal crush fracture) osteoporosis, resorption indices and dynamic measurements of trabecular bone formation based on in vivo tretracycline labeling in 7.5 mm transiliac biopsies have been compared with trends in radial cortical and trabecular bone density measured with computed tomography. Positive correlations were observed between trabecular bone density trends in the radius and indices of bone formation in the ilium. These were improved when one of the two resorption indices was included with a formation index in bivariate regressions. Marked interindividual variations in radial bone density trends were also seen in cortical bone. These correlated poorly with trends in trabecular bone. Weak negative relationships between cortical bone trends and indices relating to bone formation and resorption were observed, but a positive association was seen with single-labeled surfaces on iliac trabeculae. If, as has been suggested, there are periodic variations in bone formation, the results suggest that axial and peripheral trabecular bone density trends are synchronized in osteoporosis, perhaps in response to systemic factors, such as circulating hormones.
Interleukin-3-dependent hematopoietic stem cell lines capable of osteoclast formation in vitro
Authors: Hagenaars C.E. , Haas E.W.M. Kawilarang-de , van der Kraan A.A.M. , Spooncer E. , Dexter T.M. , Nijweide P.J.
Published: February 2010

Recently we reported that the osteoclast originates from the pluripotent hematopoietic stem cell. However, a detailed analysis of the progenitor and precursor stages of the osteoclast lineage is hard to perform with primary cultures of stem cells. In the present investigation interleukin-3 (IL-3) -dependent multipotent hematopoietic stem cells lines (FDCP-mix), which have many characteristics in common with freshly isolated hematopoietic stem cells, were assayed for their osteoclast formation capacity. FDCP-mix cell lines A4, C2GM, and 15S were cocultured with periosteum-free 17-day-old fetal metatarsal bones. The effects of culture time, medium composition, and addition of WEHI-3b-conditioned medium (an unpurified IL-3 preparation) on osteoclast formation were studied. 15S cells never differentiated into osteoclasts. Both A4 and C2GM cells were able to generate osteoclasts. Osteoclast formation was visualized by staining for tartrate-resistant acid phosphatase activity and confirmed by 45Ca release assays and electron microscopic studies. Medium supplemented with fetal calf serum clearly supported osteoclast formation from A4 cells better than medium supplemented with cock serum. The difference between fetal calf serum and horse serum is generally less pronounced. C2GM cells formed osteoclasts more readily and, generally, earlier than A4 under all culture conditions. WEHI-3b-conditioned medium addition increased the numbers of osteoclasts and their resorption activity. The coculture of stripped metatarsal bones with FDCP-mix cell lines therefore offers a model system with many possibilities for the study of osteoclastogenesis and its regulation.
Role of colony-stimulating factors in osteoclast development
Authors: Takahashi Naoyuki , Udagawa Nobuyuki , Akatsu Takuhiko , Tanaka Hirofumi , Shionome Manabu , Dr. Suda Tatsuo
Published: February 2010

Effects of various colony-stimulating factors (CSFs) [interleukin-3 (IL-3), granulocyte-macrophage CSF (GM-CSF), macrophage CSF (M-CSF), and granulocyte CSF (G-CSF)] on osteoclast-like cell formation were examined in two different culture systems: the one-step mouse marrow culture system and the two-step coculture system of mouse primary osteoblastic cells with the bone marrow cells collected from the colonies that formed in the methylcellulose in the presence of the CSFs. In the one-step mouse marrow cultures, none of the CSFs stimulated the formation of tartrate-resistant acid phosphatase (TRAP, a marker enzyme of osteoclasts)-positive multinucleated cells (MNCs). Furthermore, the CSFs other than G-CSF inhibited in a dose-dependent manner the TRAP-positive MNC formation induced by 1α-25-dihydroxyvitamin D3 [1α,25-(OH)2D3]. In contrast, when marrow cells were first cultured in semisolid methylcellulose in the presence of a CSF and the recovered marrow cells from the semisolid cultures were subsequently cocultured with primary osteoblastic cells in the presence of 1α,25-(OH)2D3, numerous TRAP-positive MNCs were formed. [125I]salmon calcitonin specifically bound to TRAP-positive cells formed in this two-step culture system. Over 90% of the TRAP-positive mononuclear cells and MNCs accumulated [125I]calcitonin. M-CSF was the most potent in inducing TRAP-positive MNCs, followed by GM-CSF, IL-3, and G-CSF in that order. No TRAP-positive cells were formed in the absence of either osteoblastic cells or 1α,25-(OH)2D3. These results clearly indicate that the CSFs, especially M-CSF, stimulate the growth of osteoclast progenitors, which differentiate into osteoclast-like cells in response to 1α,25-(OH)2D3 in the presence of osteoblastic cells.
BM 21.0955, a potent new bisphosphonate to inhibit bone resorption
Authors: Mühlbauer R.C. , Bauss F. , Schenk R. , Janner M. , Bosies E. , Strein K. , Fleisch H.
Published: February 2010

A total of 300 new bisphosphonates were screened for their effect on bone resorption in the rat. Among these, 1-hydroxy-3-(methylpentylamino)propylidenebisphosphonate (BM 21.0955) was selected for detailed investigation. It inhibited arotinoid-stimulated bone resorption as assessed by calcemia in thyroparathyroid-ectomized rats at a SC dose as low as 0.001 mg P (0.016 μmol) per kg body weight per day. The compound was thus about 2, 10, 50, and 500 times more potent than risedronate, alendronate, pamidronate, and clodronate, respectively. Intravenous administration was as effective as subcutaneous, and oral administration was 100 times less effective. The effect after one administration decreased with time but was still measurable after 2 weeks. Nonstimulated bone resorption assayed by the urinary excretion of radiolabeled tetracycline from lifelong prelabeled animals was also inhibited. This effect started 3 days after a single dose and was still maximal after 7 days. Histomorphometric analysis of the tibial metaphysis in growing intact rats also showed an inhibition of bone resorption along with an increase in bone mass. The number of osteoclasts increased in animals treated with 0.01 and 0.1 mg P per kg (0.16 and 1.6 μmol/kg) body weight SC but decreased in animals given 1 mg P per kg (16.1 μmol/kg), showing that the inhibition of bone resorption was not due to an inhibition of osteoclast recruitment. No inhibition of mineralization occurred. This new bisphosphonate appears to have great potential for use in human bone disease.
Effect of caffeine on parameters of osteoblast growth and differentiation of a mineralized extracellular matrix in vitro
Authors: Tassinari Melissa S. , Gerstenfeld Louis C. , Stein Gary S. , Dr. Lian Jane B.
Published: February 2010

The effects of caffeine exposure on bone formation were examined using a chick osteoblast culture system. Secondary cultures of normal diploid osteoblasts were exposed to chronic doses of 0, 0.1, 0.2, or 0.4 mM caffeine beginning on day 0 through day 28. Neither the rate of cell proliferation nor cell number, as measured by total DNA, was decreased for any of the doses examined. In contrast, osteocalcin levels, alkaline phosphatase activity, and total calcium levels showed a dose-related decrease in cultures treated with caffeine. These parameters were significantly decreased at the highest dose of 0.4 mM. The reduction in total protein levels ranged rom 29 to 66% of control values and was independent of dose. In contrast, total collagen levels were more affected by the dose of caffeine used. Inhibition of collagen levels was most apparent on days 17 and 21, time points during the period of active formation of the matrix immediately preceding the deposition of mineral. By day 28 collagen levels in cultures exposed to the lower doses of caffeine had returned to control levels, and only the cultures exposed to the highest dose (0.4 mM) remained significantly inhibited with respect to both collagen and mineral. Histochemically, alkaline phosphatase and mineral staining of day 28 cultures mirrored the biochemical events with the 0.4 mM caffeine exposure. The results indicate that one of the effects of caffeine on bone development is to inhibit the formation of a competent extracellular matrix during the osteoblast differentiation sequence, which results in the inhibition of mineralization analogous to the delayed ossification observed in fetal animals after prenatal caffeine exposure.
Polyarginine, polylysine, and protamine mimic the effects of high extracellular calcium concentrations on dispersed bovine parathyroid cells
Authors: Brown Edward M. , Katz Craig , Butters Robert , Kifor Olga
Published: February 2010

We investigated the effects of the basic peptides polyarginine, protamine, and polylysine on dispersed bovine parathyroid cells. All three peptides produced a dose-dependent inhibition of dopamine-stimulated cAMP accumulation, with half-maximal inhibition at 4 × 10−8, 1.5 × 10−7, 3 × 10−7, and 2 × 10−6 M, respectively, for polyarginine, protamine, and two preparations of polylysine of molecular weights 10,200 and 3800. The inhibition of cAMP accumulation was reversible and was blocked by preincubating the cells overnight with 0.5 μg/ml of pertussis toxin. The same peptides also inhibited PTH release at similar concentrations, markedly stimulated the accumulation of inositol phosphates at two- to threefold higher concentrations, and produced transient increases in the cytosolic Ca2+ concentration (Cai) in fura-2-loaded parathyroid cells. The polylysine-evoked spike in Cai persisted despite the removal of extracellular Ca2+, indicating that it arose from intracellular Ca2+ stores. Exposure of the cells to elevated extracellular magnesium (Mg2+) concentrations elicited a similar spike in Cai but blocked the Cai transient in response to subsequent addition of polylysine, or vice versa. Thus, Mg2+ and polylysine mobilize Ca2+ from the same intracellular store(s). These results indicate that highly basic peptides closely mimic the effects of polyvalent cations on parathyroid function, suggesting that both agents may regulate parathyroid function via similar biochemical pathways.
Effect of 1,25-dihydroxyvitamin D3 on prostaglandin E2 production in cultured mouse parietal bones
Authors: Klein-Nulend Jenneke , Pilbeam Carol C. , Dr. Raisz Lawrence G.
Published: February 2010

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] was tested for its effects on prostaglandin E2 (PGE2) production and bone resorption in cultured mouse parietal bones. We found that at 24 h 1,25-(OH)2D3 increased 45Ca release but did not affect PGE2 production. However, at 48 h 1,25-(OH)2D3 produced a dose-related increase in PGE2 production. PGE2 production was increased with 1,25-(OH)2D3 at 10−10-10−8 M, and 45Ca release was increased with 1,25-(OH)2D3 at 10−11-10−8 M. The effects of 1,25-(OH)2D3 on PGE2 production persisted in the presence of cortisol (10−8 M), and the effects were greater in the presence of arachidonic acid (10−5 M) or fetal bovine serum (10%). Human interleukin-1α (IL-1, 1 ng/ml) and bovine parathyroid hormone-(1–34) (PTH, 10 ng/ml) increased PGE2 production earlier and to a greater extent than 1,25-(OH)2D3. The PGE2 response to IL-1 and PTH was not affected by 1,25-(OH)2D3 at 24 h, but at 48 h 1,25-(OH)2D3 (10−8 M) increased the PGE2 response to both IL-1 and PTH. The stimulation of 45Ca release at 48 h by high concentrations of 1,25-(OH)2D3, PTH, or IL-1 was similar, and there was no evidence for an additive effect. To test for an effect of 1,25-(OH)2D3 on endogenous IL-1 production, experiments were performed in the presence of an IL-1 receptor antagonist (IL-1Ra, 1000 ng/ml), which has been found to block selectively IL-1 effects on bone resorption and PG production. We found that IL-1Ra blocked the stimulatory effect of 1,25-(OH)2D3 (10−8 M) on PGE2 production but not on 45Ca release. We conclude that 1,25-(OH)2D3 at high concentrations is a stimulator of PGE2 release from bone and can enhance the response to PTH and IL-1. Its effect is smaller and occurs later than that of PTH and IL-1. The effect of 1,25-(OH)2D3 on PGE2 production could be mediated by endogenous IL-1.
No evidence for an effect of lactase deficiency on bone mass in pre- or postmenopausal women
Authors: Dr. Slemenda Charles W. , Christian Joe C. , Hui Siu , Fitzgerald Joseph , Johnston Conrad C.
Published: February 2010

The potential role for lactase deficiency in the development of low bone mass was examined in 342 adult female twins. Diminished lactase activity, defined as greater than 20 ppm increase in expired hydrogen at 2 or 2.5 h after an oral lactose load, was examined: (1) by comparing bone mass between members of twin pairs discordant for lactase activity; (2) by examining the linear association between bone mass and total expired hydrogen gas; and (3) by comparing all lactase-deficient individuals to those with persistent lactase activity. Among members of discordant (primarily DZ) pairs, the lactase-deficient member had greater bone mass 54% of the time. The correlations between the increase in expired hydrogen and bone mass at various sites were between −0.02 (femoral neck) and 0.11 (midshaft radius), suggesting no association between these variables. Finally, all lactase-deficient subjects were compared with those with normal lactase activity, regardless of twin status, and at each skeletal site the differences in bone mass were 1% or less. Thus, all primary hypotheses were not supported by these data; that is, in this large sample we could find no evidence of a detrimental effect of lactase deficiency on adult bone mass. However, baseline expired hydrogen was consistently and positively associated with bone mass at all sites, independently of age, suggesting the possibility that some aspect of intestinal function related to the activity of bacterial anaerobes may be positively associated with bone mass.