bluuebunny/crossref_metadata_2025 huggingface dataset:
Normalization of spine densitometry
Authors: Mazess Richard B. , Barden Howard , Mautalen Carlos , Vega Eduardo
Published: February 2010

We investigated several transformations of bone mineral content (BMC) and area density (BMD), in particular volumetric density (BMAD), to ascertain the influence on (1) body size dependence, (2) diagnostic sensitivity, and (3) precision. These transformations were examined in a group of 657 normal postmenopausal women and 327 women with osteoporotic fracture. First, expression of results as BMAD removed some of the slight dependence on body size; 21% of the variation in BMC and 15% of the variation in BMD were associated with body weight, but only 8% with BMAD. Second, the Z scores compared with those for age-matched controls for BMD and BMC were −1.85 and −1.71, respectively; the Z score for BMAD was −1.64. Third, the precision error for BMC was reduced by expressing results as BMD (1.1 versus 0.5%); BMAD degraded precision slightly (0.7%). BMD appeared to be the optimal expression for bone densitometry because it provided the best diagnostic sensitivity and lowest precision error; there was a minimal influence of body size on BMD results. This study also showed that osteoporotic women, even in the first postmenopausal decade, had low spine BMD, small vertebral area, and low body weight. Such women may be particularly at risk of crush fracture.
Effects of epidermal growth factor on parathyroid hormone-related protein production by mammary epithelial cells
Authors: Ferrari S.L. , Rizzoli R. , Bonjour J.-P.
Published: February 2010

Parathyroid hormone-related protein (PTHrP) plays a prominent role in the pathogenesis of humoral hypercalcemia of malignancy. However, it is also expressed in various nonmalignant tissues, particularly during fetal organogenesis and tissue differentiation. Thus, PTHrP is synthesized in skin, placenta, and mammary gland during lactation. Little is known, however, about the regulation of PTHrP synthesis and release in nontumoral cells. We investigated the regulation of PTHrP production by epidermal growth factor (EGF), a factor of major importance in the development of lactating breast, in primary cultures of rat mammary epithelial cells. EGF stimulated the production of immunoreactive and bioactive PTHrP in a time- and concentration-dependent manner. A 12 h incubation with 10 ng/ml of EGF increased PTHrP production by 36.0 ± 7.1% (n = 7 experiments, p < 0.01). This was accompanied by an increase in PTHrP mRNA steady-state levels. The production of PTHrP was stimulated by the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA) by 82.9 ± 9.7% (n = 4 experiments, p < 0.01). The effects of PMA and EGF were additive. The EGF-induced stimulation appeared to be independent of extracellular calcium concentration, prostaglandin, or cAMP synthesis, but may have involved tyrosine kinase-mediated mechanisms. These results indicate that EGF was capable of increasing the production of PTHrP by cultured mammary epithelial cells. They also suggest that factors activating the PKC pathway are involved in the upregulation of PTHrP expression in mammary epithelial cells.
Prediction of hip fractures from pelvic radiographs: The study of osteoporotic fractures
Authors: Glüer Claus-C. , Cummings Steven R. , Pressman Alice , Li Jiao , Glüer Kathrin , Faulkner Kenneth G. , Grampp Stephan , Genant Harry K.
Published: February 2010

To determine whether simple measurements made on conventional radiographs of the hip could predict hip fractures, we obtained pelvic radiographs on 9704 white women age 65 or older. We analyzed the radiographs of all 162 women who subsequently suffered a hip fracture and 162 randomly selected women who did not. Adjusting for age, four measurements independently predicted hip fracture: reduced thickness of the femoral shaft cortex (odds ratio 1.7 per standard deviation; 95% confidence interval 1.2, 2.3) and of the femoral neck cortex (1.4 per standard deviation; 1.0, 1.9), reduction in an index of tensile trabeculae (2.0 per unit; 1.4, 2.9), and wider trochanteric region (1.4 per standard deviation; 1.0, 2.0). The combination of these four measurements predicted hip fracture at least as strongly as did measurement of bone density of the femoral neck (areas of the receiver-operating characteristic curve = 0.81 and 0.80, respectively). We conclude that simple measurements made on pelvic radiographs predict hip fractures as well as bone density of the hip.
Picomolar norethindrone in vitro stimulates the cell proliferation and activity of a human osteosarcoma cell line and increases bone collagen synthesis without an effect on bone resorption
Authors: Lau William K.-H. , Wang Shu Ping , Linkhart Thomas A. , Demarest Keith T. , Baylink David J.
Published: February 2010

To determine how progestins increase bone formation in vivo, the effects of the synthetic progestin norethindrone (NET), on aspects of bone formation in vitro were determined. NET at picomolar concentrations in vitro stimulated the proliferation of human TE85 osteosarcoma cells as assessed by the increase in [3H]thymidine incorporation into DNA and in cell number and also stimulated the release of osteocalcin in both the presence and absence of 10 nM 1,25-(OH)2D3. NET increased cellular alkaline phosphatase specific activity (an index of osteoblastic differentiation), but at much higher concentrations, that is, nanomolar. These findings suggest that low concentrations of NET act directly on human TE85 osteosarcoma cells to stimulate their proliferation, differentiation, and cell activity. Furthermore, mitogenic doses of NET stimulated bone collagen synthesis both in a chicken calvarial organ culture assay (assessed by the incorporation and hydroxylation of [3H]proline) and in a human TE85 osteosarcoma cell culture assay (determined by the incorporation of [3H]proline into collagenase-digestible proteins). In contrast, NET at 10−6-10−12 M had no apparent effect on the rate of basal or PTH-stimulated release of 45Ca from prelabeled mouse calvariae in vitro. In summary, this study has demonstrated for the first time that picomolar NET acted directly on human TE85 osteosarcoma cells to increase (1) cell proliferation and differentiation, (2) osteoblastic activity (i.e., osteocalcin synthesis), and (3) bone collagen synthesis in vitro. The same doses of NET in vitro did not reduce the bone resorption rate under our assay conditions. In conclusion, these in vitro osteogenic activities of NET suggest that it may be a useful therapeutic agent to increase bone formation in patients with low bone mass.
Calcium signaling in endothelin- and platelet-derived growth factor-stimulated chondrocytes
Authors: Dr. Stojilkovic Stanko S. , Vukicevic Slobodan , Luyten Frank P.
Published: February 2010

In bovine articular chondrocytes, endothelin (ET) and platelet-derived growth factor (PDGF) receptors mediate agonist-induced increases in inositol phosphate hydrolysis, cytoplasmic calcium concentration ([Ca2+]i), and mitogenesis. In most cells, ET stimulated nonoscillatory [Ca2+]i elevations with dose-dependent increases in both spike and plateau amplitudes. However, about 15% of cells showed oscillatory Ca2+ responses with a constant frequency and variable shape and duration of spiking. ET-1 and ET-2 were more potent than ET-3 in stimulating [Ca2+]i responses in inhibiting the specific binding of 125I-ET-1 and 125I-ET-3 and in promoting internalization of the receptor-ligand complex, consistent with actions through endothelin ETA receptors. Similar nonoscillatory and oscillatory patterns of Ca2+ responses were observed in PDGF-stimulated cells. In cells showing nonoscillatory Ca2+ responses to ET-1, subsequent stimulation with PDGF was frequently followed by the development of an oscillatory Ca2+ response. Nonoscillatory responses to both agonists were only slightly reduced in Ca2+-deficient medium, but the oscillatory responses were critically dependent on Ca2+ entry. Ca2+ spiking was not altered in the presence of the voltage-sensitive Ca2+ channel blocker, nifedipine; also, depolarization of chondrocytes by high K+ did not induce [Ca2+]i responses, confirming that voltagesensitive calcium channels are not expressed in these cells. At high agonist concentrations, ET- but not PDGF-stimulated cells underwent rapid desensitization. Activation of ETA and PDGF receptors was associated with differential stimulation of thymidine incorporation; ET-1 induced a low-amplitude bell-shaped doseresponse curve; PDGF induced a sustained sigmoidal and dose-dependent rise. These data indicate that two distinct types of Ca2+-mobilizing receptors initiate similar patterns of [Ca2+]i responses but have different capacities to maintain and reinitiate the Ca2+ signaling, as well as to promote mitogenesis.
Opposite effects of osteogenic protein and transforming growth factor β on chondrogenesis in cultured long bone rudiments
Authors: Dieudonné S.C. , Semeins C.M. , Goei S.W. , Vukicevic S. , Nulend Klein J. , Sampath T.K. , Helder M. , Burger E.H.
Published: February 2010

Osteogenic protein-1 (OP-1, also called BMP-7) is a bone morphogenetic member of the TGF-β superfamily. In the present study, we examined the effect of recombinant human OP-1 on cartilage and bone formation in organ cultures of metatarsal long bones of mouse embryos and compared the OP-1 effects with those of human TGF-β1 and porcine TGF-β1 and β2. Cartilage formation was determined by measurement of longitudinal growth of whole bone rudiments during culture and by the incorporation of 35SO4 into glycosaminoglycans. Mineralization was monitored by 45Ca incorporation in the acid-soluble fraction and by measuring the length of the calcifying center of the rudiment. Toluidine blue-stained histologic sections were used for quantitative histomorphometric analysis. We found that OP-1 stimulated cartilage growth as determined by sulfate incorporation and that it increased remarkably the width of the long bones ends compared with controls. This effect was partly caused by differentiation of perichondrial cells into chondrocytes, resulting in increased appositional growth. In contrast to OP-1, TGF-β1 and β2 inhibited cartilage growth and reduced the length of whole bone rudiments compared with controls. In the ossifying center of the bone rudiments, both OP-1 and TGF-β inhibited cartilage hypertrophy, growth of the bone collar, and matrix mineralization. These data demonstrate that OP-1 and TGF-β exhibit opposite effects on cartilage growth but similar effects on osteogenesis in embryonic mouse long bone cultures. Since both OP-1 and TGF-β have been demonstrated in embryonic cartilage and bone, these results suggest that they act as autocrine or paracrine regulators of embryonic bone development.
Effects of transforming growth factor β on bone nodule formation and expression of bone morphogenetic protein 2, osteocalcin, osteopontin, alkaline phosphatase, and type I collagen mRNA in long-term cultures of fetal rat calvarial osteoblasts
Authors: Harris S.E. , Bonewald L.F. , Harris M.A. , Sabatini M. , Dallas S. , Feng J.Q. , Ghosh-Choudhury N. , Wozney J. , Mundy G.R.
Published: February 2010

Transforming growth factor β (TGF-β) is one of the most abundant of the known growth regulatory factors stored within the bone matrix. When bone is resorbed, TGF-β is released in an active form and is a powerful bone growth stimulant. When injected into the subcutaneous tissue over the calvarial surface of rodents, it rapidly causes proliferation of the periosteal layer and accumulation of new woven bone. In this report, we describe the effects of TGF-β1 on first subcultures of fetal rat osteoblasts obtained from calvarial bones and cultured from confluence with ascorbic acid and β-glycerophosphate. Under these conditions, nodules with characteristics of normal bone appear by day 8. Similar to experiments described by Antosz et al., TGF-β added to confluent cultures inhibited the formation of bone nodules. Both the number and total area of the nodules were quantitated and shown to be completely inhibited by 2 ng/ml of TGF-β1. TGF-β also impaired the expression of genes associated with bone formation, including type I collagen, alkaline phosphatase, osteopontin, and osteocalcin. TGF-β also inhibited the expression of mRNA for the bone morphogenetic protein 2 (BMP-2). These results, showing suppression of markers representative of osteoblast differentiation, suggest that the effects of TGF-β to stimulate bone formation in vivo are not likely a result of effects on differentiated mineralizing osteoblasts but, as suggested by previous studies, more likely are caused by effects on osteoblast precursors. These results also suggest that endogenous BMP-2 expression in fetal rat calvaria cells is important for bone cell differentiation.
Anabolic effect of high doses of human parathyroid hormone (1–38) in mature intact female rats
Authors: Jerome C.P.
Published: February 2010

Adult female retired breeder Wistar rats were treated with placebo or with synthetic human parathyroid hormone (hPTH) fragment (1–38) by subcutaneous injection at doses ranging from 50 to 1000 μg/kg, 5 days per week, for 2–8 weeks. One group of rats received hPTH-(1–38), 400 μg/kg, 5 days per week for 4 weeks, followed by 4 weeks placebo. The effects on bone mass and structure were evaluated by histomorphometric analysis of cross sections of femoral metaphyses. The effects on bone formation were evaluated by measuring uptake of [H]tetracycline into femora and by qualitative evaluation of histologic sections after labeling with calcein and tetracycline. All doses of hPTH-(1–38) were anabolic. Lamellar bone formation was stimulated on all surfaces, and cancellous bone mass was increased as a result of trabecular thickening. Small quantities of woven bone were also formed in marrow cavities after treatment with 400 or 1000 μg/kg. Treatment with 1000 μg/kg had an effect on measured parameters similar to that with 400 μg/kg, and there was no evidence for a diminution of the anabolic effect at high doses. Animals treated with hPTH-(1–38) for 4 weeks followed by 4 weeks placebo tended to have less bone than those that received hPTH-(1–38) for 8 weeks but had significantly more bone than placebo-treated rats. In conclusion, daily injection of hPTH-(1–38) for 2–8 weeks at doses ranging from 50 to 1000 μg/kg had an anabolic effect on both cortical and trabecular bone in the femur of mature female intact rats. After withdrawal of treatment, the bone formation rate fell below levels in untreated animals, but bone mass remained higher.
New model for bone resorption study in vitro: Human osteoclast-like cells from giant cell tumors of bone
Authors: Grano Maria , Colucci Silvia , De Bellis Michele , Zigrino Paola , Argentino Luigi , Zambonin Giovanni , Serra Massimo , Scotlandi Katia , Teti Anna , Prof. Zallone Alberta Zambonin
Published: February 2010

Cells harvested from 12 human giant cell tumors of bone and kept in culture for several passages were characterized for bone-resorbing capability, total and tartrate-resistant acid phosphatase activity, response to the calciotropic hormone calcitonin, cell proliferation, multinucleation after passages, and presence of calcium sensing. Cells obtained from three tumors presented a complete panel of osteoclast characteristics and maintained their multinuclearity after several passages. Cells from four other tumors increased their cAMP levels after treatment with calcitonin, and the other five apparently consisted of cells of stromal origin. These human cell populations with osteoclast characteristics may provide valid in vitro models for the investigation of osteoclastic differentiation and activity.
Weight-bearing activity during youth is a more important factor for peak bone mass than calcium intake
Authors: Welten D.C. , Prof. Dr Kemper H. C. G. , Post G.B. , van Mechelen W. , Twisk J. , Lips P. , Teule G.J.
Published: February 2010

In a 15 year longitudinal study (Amsterdam Growth and Health Study) is evaluated the effect of daily calcium intake (CAI) during adolescence and young adulthood on the development of peak bone mass at age 27 when the influence of weight-bearing activity (WBA) and body weight was accounted for. A group of 84 males and 98 females were measured longitudinally from age 13 until age 28. Measurements were taken six times of anthropometric characteristics. Lifestyle was also evaluated six times by cross-check interviews of CAI and WBA. Bone mineral density (BMD) of the lumbar spine was determined at age 27 by dual x-ray absorption. Three periods were considered, that is, the adolescent period, the period 13–21 years, and the total period (13–27 years). In multiple linear regression analyses, only WBA and body weight were significant positive contributors to the final model of lumbar BMD at age 27. In all three periods WBA was the best predictor in males and body weight in females. ANOVA was performed on BMD and the highest and lowest quartiles of calcium intake with the significant predictor variables of the linear regression model as covariates. Again calcium intake appeared not a significant predictor of BMD in the three periods in both sexes. Regular weight-bearing exercise and at least a normal age-related body weight in adolescence and young adulthood are of key importance in reaching the highest lumbar peak bone mass at the age of 27 years.
Reference data for bone mass, calciotropic hormones, and biochemical markers of bone remodeling in older (55-75) postmenopausal white and black women
Authors: Kleerekoper Michael , Nelson Dorothy A. , Peterson Edward L. , Flynn Michael J. , Pawluszka Annie S. , Jacobsen Gordon , Wilson Paulette
Published: February 2010

From a random sample of our institution's health maintenance organization (HMO), we recruited 250 white women and 112 black women, aged 55-75, all of whom were 10 or more years postmenopause with minimal estrogen exposure and free of osteoporosis, other metabolic bone disease, and medical, surgical, or therapeutic situations that may influence bone loss. Bone mass was measured in the radius, spine, and femur by DXA and in L1 by QCT. Serum samples were analyzed for parathyroid hormone, calcidiol, calcitriol, osteocalcin, and bone alkaline phosphatase and urine samples analyzed for creatinine, calcium, and hydroxyproline. Mean Z score, based on published reference data for forearm and femoral neck BMD in the white women, was not significantly different from zero, but mean Z score at the lumbar spine was 0.6 (p > 0.001), 17.2% of the individual values being >2.0. In normal white women (BMI > 27.3, n = 143), Z score was still >2.0 in 10.3%, suggesting that the upper bound of the published reference interval may be too low. After adjustment for body mass index, BMD was greater in the forearm (9.8%), spine (8.7%), and femoral neck (14.7%) in black women (p > 0.001 at all sites). At L1, adjusted BMC in the black women was 37.4% greater than in the white women (p > 0.001). Serum calcidiol was significantly lower and serum PTH and calcitriol significantly higher in the black women. Despite this, biochemical markers of bone resorption and formation were significantly lower in the black women. We conclude that skeletally healthy older black women have a greater bone mass and lower rates of bone remodeling than a comparable group of white women. These data can serve as reference intervals for the variables measured.
Profile of ligand specificity of the vitamin D binding protein for 1α,25-dihydroxyvitamin d3 and its analogs
Authors: Bishop June E. , Collins Elaine D. , Okamura William H. , Professor Norman Anthony W.
Published: February 2010

The profile of structural preference for the ligand binding domain of the human vitamin D binding protein (DBP) was determined by steroid competition assay of 71 analogs of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. The following categories of structural modification were evaluated [values represent fold change; R = reduction, I = increase in binding to the DBP from the reference 1α,25(OH)2D3]: (1) deletion in the A ring of the 1α-hydroxyl (20-1600I); (2) conversion of the triene system to the previtamin form (6-40R); (3) addition of substituents to carbon 11 of the C ring (4-14R); (4) inversion of the C/D ring junction (8-20R); (5) unsaturation of the D ring (16-ene; 4-140R); (6) replacement of hydrogen with deuterium atoms (no effect); alteration of the side chain by (7) adding or deleting carbon atoms (5-12R); (8) addition of fluorines (0.2-10R); (9) presence of unsaturation (22-ene, 0-5R; 23-ene, 3R-10I; 23-yne, 5-20R); (10) addition of hydroxyls (2-100R); and (11) addition of an aromatic ring (0-20I). Thus the DBP ligand binding domain could tolerate only modest changes to the structure of 1α,25(OH)2D3 without a reduction in binding of the analog. The increases in binding seen in the aromatic side chain and with a triple bond at carbon-23 may be indicative of a preferred conformation of the flexible 1α,25(OH)2D3 side chain. In addition, a comparison was made of the DBP ligand binding domain with that of the human HL-60 cell 1α,25(OH)2D3 nuclear receptor. Both ligand binding domains could equivalently accommodate to the presence of (1) a side-chain cyclopropyl group, (2) 22-ene or 23-yne, (3) lengthening the side chain by two carbons, (4) presence of four to six fluorine atoms, (5) substitution of an oxygen for carbon 22, and (6) presence of a 22-[m-(dimethylhydroxymethyl)phenyl] aromatic group in the side chain. The DPB could tolerate better than the HL-60 cell receptor the presence of a 22-(p-hydroxyphenyl) aromatic group in the side chain and the absence of the 1α-hydroxyl. In contrast, the HL-60 cell receptor could tolerate better than the DBP the following structural modifications: presence of a 16-ene, or 16-ene plus 23-yne unsaturation, and presence of an 11β-hydroxyl.
Comparison of dosage-dependent effects of β-aminopropionitrile, sodium fluoride, and hydrocortisone on selected physical properties of cortical bone
Authors: DR. Lees Sidney , Hanson Douglas , Page Elizabeth , Mook Herbert A.
Published: February 2010

BAPN, sodium fluoride, and hydrocortisone are reported to induce altered mineralization states. Three separate sets of experiments, one set for each agent, were performed using male New Zealand white rabbits. In each experiment the rabbits were segregated into groups, each fed a specified weight-determined dose for 13 weeks and then sacrificed. Compact bone from the left femur and tibia were tested for density, composition, sonic velocity, longitudinal elastic modulus, equatorial diffraction spacing of mineralized collagen, diaphyseal cross-sectional area, and relative load stress. β-Aminopropionitrile (BAPN) induced monotonic degradation of all properties at all dose levels, corresponding to the decreasing density with dosage level. The elastic moduli show a decrease; the equatorial diffraction spacing of the collagen increases. The cross-sectioned diaphysis resembled woven bone. The variability in properties increased with dosage. The total cross-sectional area for a given weight increased, implying that the decreased elastic properties were compensated for by a larger area to support the weight. There was a slight increase in average density and other properties for fluoride-treated rabbits, peaking at 20 mg/kg BW/day. For higher dosages the properties are degraded and the values were much lower at high fluoride dosages than for BAPN. There was no peak for the equatorial diffraction spacing, which increased with dosage. It is inferred that the fluorosed apatite is denser than normal apatitic mineral and therefore has a smaller specific volume. A greater weight fraction of fluorosed mineral has a smaller volume fraction than the equivalent normal apatitic mineral. The bone sections look more normal, except for the porosis. The total cross-sectional area decreases when the bone density increases and then increases as the density falls, again implying that the area required to support body weight depends on the magnitude of the elastic moduli. There was a small change in some of the properties of the bones of the hydrocortisone-treated rabbits, but the effects on others were undetectable within the uncertainty of the procedures. There was no change in the cross-sectional areas of the diaphyses.
Does estimating volumetric bone density of the femoral neck improve the prediction of hip fracture? A prospective study
Authors: Cummings Steven R. , Marcus Robert , Palermo Lisa , Ensrud Kristine E. , Genant Harry K.
Published: February 2010

Standard projectional bone density of the femoral neck (BMD), defined as bone mineral content divided by the projected area of the neck, predicts hip fractures but may not accurately estimate the true volumetric bone density of the femoral neck. To determine whether an estimate of the volumetric bone density of the neck, “bone mineral apparent density” (BMAD), would be a better predictor of hip fracture, we analyzed dual x-ray absorptiometry scans obtained prospectively from 7963 older white women, of whom 83 suffered a hip fracture during follow-up. Both BMD and BMAD were stronger predictors than bone mineral content (BMC) of the femoral neck. However, BMD and BMAD had very similar predictive values for hip fracture: each standard deviation decrease in either BMD or BMAD of the femoral neck increased the age-adjusted risk of hip fracture 2.6- to 2.7-fold. We conclude that BMD and BMAD of the femoral neck have a similarly strong predictive value for hip fracture.
Urinary hydroxypyridinium crosslinks of collagen in population-based screening for overt vertebral osteoporosis: Results of a pilot study
Authors: Seibel M.J. , Woitge H. , Scheidt-Nave C. , Leidig-Bruckner G. , Duncan A. , Nicol P. , Ziegler R. , Robins S.P.
Published: February 2010

The urinary pyridinium crosslinks pyridinoline (PYD) and deoxypyridinoline (DPD) have been shown to provide valid indices of bone resorption. At present, both crosslink components are determined by reversed-phase HPLC, a time-consuming method precluding the use of these markers for routine purposes. Therefore, efforts have been made to develop simple immunoassays for the rapid measurement of urinary crosslinks, and their application to large-scale osteoporosis screening has been proposed. To evaluate the applicability and diagnostic validity of pyridinium crosslink measurements for screening purposes, urinary concentrations of total and free PYD and DPD were determined by HPLC and immunoassay technique (ELISA) in a sample of 269 individuals (male to female ratio = 130:139; age 50-81 years) recruited at random within a population survey of vertebral osteoporosis. On a molar basis, ELISA measures of crosslink-related epitopes were highly correlated with both total and free PYD and DPD as determined by HPLC (r > 0.82, p < 0.001). Age-specific means for creatinine-corrected total and free pyridinium crosslinks were significantly higher in females than in males (p < 0.001). In both sexes, neither age nor anthropometric variables (weight, height, and body mass index) showed a linear effect on the urinary crosslink/creatinine ratio. On average, 50% of the total amount of urinary crosslinks were present in free form. For both PYD and DPD, this proportion was significantly higher in women than in men (p < 0.05), but no change was observed with age or anthropometric measures. The excretion of pyridinium crosslinks was higher in osteoporotic (n = 18) than in nonosteoporotic individuals (n = 208) from the same population. However, this difference was statistically significant (p < 0.05) only for total PYD, total DPD, and free DPD as determined by HPLC, whereas no significant difference was seen with the ELISA. However, ROC analysis showed none of the crosslink measurements to provide sufficient diagnostic validity for screening purposes with regard to vertebral osteoporosis. Our results demonstrate that (1) the immunoassay technique provides measurements of pyridinium crosslinks that are highly correlated with HPLC findings; (2) beyond age 50, urinary concentrations of total and free pyridinium crosslinks are not linearly influenced by age or anthropometric factors but change with sex independently of osteoporotic status; and (3) although pyridinium crosslink excretion provides a valid and clinically useful measure of bone resorption, a single determination lacks the sensitivity necessary to screen successfully for vertebral osteoporotic fractures.
Improved method for analysis of whole bone torsion tests
Authors: Levenston Marc E. , Beaupré Gary S. , van der Meulen Marjolein C. H.
Published: February 2010

Structural tests, such as whole bone torsion tests, have become widely accepted methods for assessing average bone material properties. To simplify interpretation of these tests, the nonuniform bone geometry is often analyzed as a tube with a constant cross section (prismatic) and the areal properties of the smallest bone section. This approach may not adequately represent the true torsional behavior of the cross section and does not account for any lengthwise variations in bone geometry. The errors introduced by these approximations are particularly significant when comparing bones of different sizes and geometries. In this paper, we examine the effects of approximating the cross-sectional torsional behavior and of neglecting lengthwise variations in bone geometry. We then present a simple, standardized procedure utilizing a FORTRAN computer program for accurate determination of material properties. We examine first simple idealized bone geometries and then a complex three-dimensional model of the femur from a 26-day-old male Sprague-Dawley rat. For these models, the conventional methods for interpreting torsion tests introduce errors of up to 42% in the shear modulus and up to 48% in the maximum shear stress; a straightforward extension of these methods reduces the errors to within 3%.
Regulation of protein kinase C by transforming growth factor β1 in rat costochondral chondrocyte cultures
Authors: Sylvia V.L. , Mackey S. , Schwartz Z. , Schuman L. , Gomez R. , Boyan B.D.
Published: February 2010

Transforming growth factor β (TGF-β) regulates the proliferation and differentiation of chondrocytes; however, the mechanism of TGF-β signal transduction remains unclear. We examined whether the response to TGF-β is mediated by protein kinase C activity in chondrocytes at different stages of maturation. The aims were to examine the effect of recombinant human TGF-β1 (rhTGF-β1) on protein kinase C in rat costochondral chondrocyte cultures; determine the major isoform present; assess the involvement of phospholipase C or tyrosine kinases; determine whether genomic or nongenomic pathways are involved; and test whether these mechanisms differ as a function of the stage of cell maturation. Dose-dependent increases in protein kinase C activity were observed in confluent, fourth-passage cultures of rat costochondral growth zone and resting zone chondrocytes treated with rhTGF-β1. In growth zone cells, elevated activity was observed at 12 h and decreased markedly by 24 h. In resting zone cells, elevated activity was observed at 9 h, maximum stimulation occurred at 12 h, and activity returned to baseline levels after 48 h. Immunoprecipitation studies showed protein kinase C α is the major isoform present in both untreated and treated cells. Neither the phospholipase C inhibitor, U73122, nor the tyrosine kinase inhibitor, genistein, significantly reduced the protein kinase C response to rhTGF-β1. Actinomycin D and cycloheximide, inhibitors of transcription and translation, produced dose-dependent inhibition of rhTGF-β1 stimulated protein kinase C activity in both resting zone and growth zone chondrocytes. The time course of activation and insensitivity to U73122 suggest that phospholipase C-mediated events are not involved in rhTGF-β1 stimulation of protein kinase C in costochondral chondrocytes. Similarly, because genistein had no effect, tyrosine kinases are not implicated. Rather, the reduction in protein kinase C activity observed when rhTGF-β1 is administered along with actinomycin D or cycloheximide indicates that new gene expression and protein synthesis are required for the response. These results indicate that the effect of rhTGF-β1 is mediated by protein kinase C; however, it is very slow and may require new protein kinase C production, perhaps via a cytokine cascade. Moreover, the classic mechanism of activation of protein kinase C by phospholipase C was not found, suggesting a novel mechanism of activation. Finally, the effects of rhTGF-β1 on protein kinase C are dependent on the state of cell maturation with respect to onset and duration of response.
In situ hybridization to show sequential expression of osteoblast gene markers during bone formation in vivo
Authors: Zhou H. , Choong P. , McCarthy R. , Chou S.T. , Martin T.J. , DR. Ng Kong Wah
Published: February 2010

We investigated the sequence of expression of osteoblast gene markers during bone formation in vivo by in situ hybridization. Cylindrical lesions were induced in the femora of sheep with titanium analytic bone implants that allow removal of serial core samples to study bone formation. At 2 weeks (2W), granulation tissue made up of spindle-shaped cells had partially replaced the blood clot. Islands of osseous tissue, first noted in the periphery of the ingrowing tissue at 3W, became the predominant tissue by 6W. The surfaces of newly forming bone at 3W were apposed by cuboidal cells, which in some areas were several layers thick. By 6W, most of the cells lining bone trabeculae had assumed a flattened morphology. The temporal and spatial distribution of osteoblast gene markers was examined by in situ hybridization with nonradioactive digoxigenin probes for α1(I) procollagen, alkaline phosphatase (ALP), osteopontin (OP), and bone Gla protein (BGP). The spindle-shaped cells in the granulation tissue expressed mRNA for α1(I) procollagen, ALP, and OP but not BGP, suggesting that they may be osteoblast precursor cells. α1(I) procollagen mRNA was strongly expressed by all cells on the surface of bone, with a peak intensity at 3W and then reducing sharply by 6W. Initially, only pockets of cuboidal cells on bone surfaces expressed ALP mRNA, with a peak intensity at 5W. Similarly, only a proportion of cuboidal cells expressed OP mRNA early in bone formation, but the number of cells expressing OP mRNA increased with time. Clumps of cuboidal cells expressed BGP mRNA only when bone was present, and the degree of expression increased with the amount of bone formed. This model allows the study of temporal and spatial sequence of gene expression in cells participating in osteogenesis. The temporal sequence is similar to that shown in vitro in other models of mineralization. The geographic localization of cells expressing mRNA for α1(I) procollagen, ALP, OP, and BGP implies subspecialization of osteoblasts in bone formation.
Universal standardization for dual X-ray absorptiometry: Patient and phantom cross-calibration results
Authors: Genant Harry K. , Grampp Stephan , Glüer Claus C. , Faulkner Kenneth G. , Jergas Michael , Engelke Klaus , Hagiwara Satoshi , van Kuijk Cornelis
Published: February 2010

The comparison of patient data among different dual x-ray absorptiometry (DXA) scanners is complicated because no universally accepted cross-calibration procedure or standard currently exists. This study was performed under the auspices of the International DXA Standardization Committee to establish appropriate cross-calibration parameters. Posteroanterior (PA) lumbar spine measurements of 100 women, ages 20–80 years (mean 52.6 + 16, range of BMD = 0.4–1.6 g/cm2) were obtained on a Norland XR26 Mark II, a Lunar DPX-L, and a Hologic QDR 2000 densitometer using standard procedures (pencil beam mode for all three scanners). Area, BMC, and BMD results from the different scanners were compared for all patients. In addition, the European spine phantom (ESP) and the European spine phantom prototype (ESP prototype), as well as standard phantoms from all three manufacturers, were evaluated on the three systems. To achieve universal scanner calibration, we used the intercept and slope of the patient's correlations and the value of the middle vertebra of the ESP as a reference point in a series of standardization formulas, and we have expressed the results as sBMD (mg/cm2). The correlations of the patients' spinal BMD values were excellent for each of the three scanner pairs. The average absolute difference in patient spinal BMD values (L2–4) between Hologic and Norland was 0.012 g/cm2 (1.3%); it was 0.113 g/cm2 (11.7%) between Hologic and Lunar and 0.118 g/cm2 (12.2%) between Norland and Lunar. The phantoms' regression lines approximated those of the patient regression lines, and the phantoms with only one measurement point were very close to the patients' regression lines. After applying the standardization formulas, the average absolute differences for the 100 patients were 28 mg/cm2 (2.7%) for Hologic/Norland, 23 mg/cm2 (2.2%) for Hologic/Lunar, and 29 mg/cm2 (2.8%) for Norland/Lunar. Average BMD results for the patients before correction were 0.972 g/cm2 for Hologic, 1.100 g/cm2 for Lunar, and 0.969 g/cm2 for Norland. After correction, sBMD results for patients were 1045 mg/cm2 for Hologic, 1047 mg/cm2 for Lunar, and 1043 mg/cm2 for Norland. The standardization approach as performed in our study provided compatibility of DXA results obtained on different scanners.
Localization of the mRNA for bone matrix proteins during fracture healing as determined by in situ hybridization
Authors: Hirakawa Kimiaki , Hirota Sehchi , Ikeda Tohru , Yamaguchi Akira , Takemura Teiji , Nagoshi Junsuke , Yoshiki Shusaku , Suda Tatsuo , Kitamura Yukihiko , Nomura Shintaro
Published: February 2010

The expression of the mRNAs for osteonectin (ON), osteopontin (OPN), osteocalcin (OC), and matrix Gla protein (MGP) was studied by in situ hybridization during the healing process of an experimental fracture in adult rat femora. At day 1 postoperatively, ON mRNA was detected in the proliferating periosteum. At day 3, ON, OPN, and OC mRNAs were detected in woven bone. From day 5, MGP and ON mRNAs were detected in the immature chondrocytes. From day 7, ON, OPN, and OC mRNAs were detected in the osteoblastic cells in newly formed endosteal trabecular bone. OPN mRNA was also detected in some of the osteocytes in trabecular bone. From day 14, OPN and MGP mRNAs were detected in newly formed periosteal hypertrophic chondrocytes, and the ON, OPN, and OC mRNAs were detected in osteoblastic cells in newly formed periosteal trabecular bone. Although the cell types that expressed each mRNA in fractured bones were similar to those in embryonic bones, the time course of these mRNA expression in fractured bones was different from that in embryonic bones. We considered that this system is useful to investigate the phenotypic change in osteogenic and chondrogenic lineage cells that appears during fracture healing at the molecular level.
Effects of free mobilization and low- to high-intensity treadmill running on the immobilization-induced bone loss in rats
Authors: Kannus Pekka , Sievänen Harri , Järvinen Teppo L.N. , Järvinen Markku , Kvist Martti , Oja Pekka , Vuori Ilkka , Jozsa Laszlo
Published: February 2010

After an immobilization period of 3 weeks, the effects of free remobilization (8 weeks) as well as low- and high-intensity treadmill running on the bone mineral content (BMC) and density (BMD) of the hindlimbs of Sprague-Dawley rats (n = 70) were studied using a dual-energy x-ray absorptiometric scanner. In the low-intensity running program, the rats were allowed to move freely in the cage for 1 week, after which they started to run on a treadmill twice a day for 7 weeks. The speed of the treadmill was 20 cm/s, with an uphill inclination of 10°. The running time was gradually increased from 20 minutes per session to 45 minutes per session. In the high-intensity group, the program was similar, with the exception that the speed of the treadmill was 30 cm/s, with an uphill inclination of 30°. Immobilization for 3 weeks produced a significant BMC and BMD loss in the immobilized left femur and tibia (mean loss 9.6%, p < 0.001) but did not affect the right free limbs. Both low- and high-intensity running restored mineral content in the immobilized limb; however, an average 5% difference (p < 0.05) in mineral content of the right and left limb bones persisted. In the running groups, the values for the immobilized left limbs were at the same level or exceeded (range 3.8–11.6%, p < 0.05–0.01) and those of the free right limbs exclusively exceeded (range 5.3–15.9%, p < 0.05–0.01) the corresponding values of the age-matched control rats. In the group with free remobilization, the BMCs and BMDs always remained below those in the controls (range –3.2 to –12.6%, p values ranging from NS to p < 0.01). Thus, this study showed the need for greater than normal activity to restore the BMC and BMD from disuse to normal.
Determinants of endogenous fecal calcium in healthy women
Authors: Heaney Robert P. , Recker Robert R.
Published: February 2010

Endogenous fecal calcium (EFC) excretion was measured in 518 studies in 191 normal perimenopausal women, most studied two to three times over a 15 year period. EFC averaged 102 + 25 mg/day. Absorption fraction was simultaneously determined by both double-isotope and balance methods. EFC was found to vary inversely with absorption fraction, and the observed relationship was used to calculate the total amount of calcium (TIC) entering the gut from endogenous sources. TIC averaged 140 + 34 mg/day and was found to be correlated with a number of intake and body size variables. Phosphorus intake was the most strongly correlated of all the variables (r = 0.404; P < 0.0001), each increment of 0.1 g phosphorus intake being associated with an increase in TIC of 6 mg. Lean body mass was the best correlated of the body size variables, with TIC rising by 1.6 mg/day for every kg lean mass. There were also small but significant correlations with protein and energy intakes, the latter suggesting that some of the variation of TIC is related to the amount of food consumed. Caffeine, previously reported as elevating TIC, did not exhibit a significant relationship in this study.
Comparison of two parathyroid hormone assays for the rat: The new immunoradiometric and the older competitive binding assay
Authors: Jara Aquiles , Bover Jordi , Lavigne Jeffrey , Felsenfeld Arnold
Published: February 2010

A competitive binding assay for parathyroid hormone (PTH INS) has been used since 1986 to measure PTH in rats. During the past year an immunoradiometric assay for the measurement of PTH (PTH IRMA) in the rat was developed. The purpose of the present study was to compare results obtained with the PTH INS and IRMA and to provide a framework for comparison for investigators who have used the PTH INS in previous studies. A total of 99 rats were studied; 27 rats had normal renal function, and 72 rats had surgically induced renal failure. In the azotemic rats, the magnitude of hyperparathyroidism was varied by changing the calcium and phosphorus composition of the diet. The correlation between the two PTH assays in the 99 rats was r = 0.98, p < 0.001. For the 27 rats with normal renal function, the correlation even within the narrow range of normal PTH values was significant, r = 0.71, p < 0.001. In the 72 azotemic rats, in which the highest INS PTH value was approximately 17 times normal, the correlation between the two PTH assays was r = 0.98, p < 0.001. The PTH IRMA provides distinct advantages, such as extended standard range, shortened incubation time, increased sensitivity, and technical simplicity, but our results indicate that the PTH INS provided an accurate measurement of PTH. Furthermore, our results should provide investigators who have used the PTH INS in previous studies with a framework for comparison with studies in which the PTH IRMA was used.
Effects of transforming growth factor β2 on the regulation of osteocalcin synthesis in human mg-63 osteosarcoma cells
Authors: Pirskanen Asta , Jääskeläinen Tuna , Mäenpää Pekka H.
Published: February 2010

Treatment of human MG-63 osteosarcoma cells with human recombinant transforming growth factor β1 (TGF-β1) was found to inhibit cell proliferation. In addition, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced osteocalcin synthesis was greatly influenced by TGF-β1. Dose- and time-dependent inhibition was seen both in medium osteocalcin and the corresponding mRNA concentrations. Furthermore, TGF-β1 decreased osteocalcin synthesis modulated negatively by dexamethasone or positively by retinoic acid. The stability of osteocalcin mRNA was not decreased by the TGF-β1 treatment, but in vitro transcription assays demonstrated diminished osteocalcin gene transcription caused by the TGF-β1 treatment. Binding of vitamin D receptor (VDR) to an oligonucleotide probe containing the osteocalcin vitamin D response element (VDRE) was not influenced by TGF-β1, however. Incubation of the cells with the serine/threonine kinase inhibitor H-7 did not block the ability of TGF-β1 to decrease osteocalcin synthesis but caused a further inhibition. Also, the 1,25(OH)2D3-induced osteocalcin synthesis was decreased by H-7 treatment, suggesting that phosphorylation as such is involved in the transcriptional activation mechanism of VDR. These results demonstrate that TGF-β1 is a strong inhibitor of the synthesis of osteocalcin, a calcium binding protein participating in bone mineralization, by counteracting the stimulatory effects of other hormones on its synthesis. We further suggest that TGF-β1 affects the synthesis of osteocalcin at the level of transcription through mechanism(s) different from the serine/threonine kinase pathway.
Platelet-activating factor induces pseudopod formation in calcitonin-treated rabbit osteoclasts
Authors: Gravel Michel R. , Zheng Zhu Gang , Sims Stephen M. , Dr. Dixon S. Jeffrey
Published: February 2010

We demonstrated previously that platelet-activating factor (PAF), a potent inflammatory mediator, acts on osteoclasts to elevate cytosolic [Ca2+] and stimulate resorption. However, it is not clear whether the effects of PAF on resorptive activity are direct or indirect. In the present study, we investigated the effects of PAF on osteoclast motility. Osteoclasts were isolated from the long bones of neonatal rabbits, and cell motility and morphology were monitored using time-lapse video microscopy. Calcitonin, a hormone known to induce retraction of pseudopods and inhibit resorptive activity, was used to render osteoclasts quiescent. Within 10 minutes of calcitonin treatment (100 ng/ml, final), pronounced retraction of pseudopods was observed in 68 of 112 cells tested. When PAF (200 nM, final) was added 10 minutes after calcitonin treatment, pseudopods were evident 1 h later in 15 of 37 calcitonin-responsive cells tested. In contrast, pseudopods were evident in only 4 of 31 calcitonin-responsive cells treated with control solutions (PAF-vehicle or S-PAF, the biologically inactive stereoisomer of PAF). Pseudopod formation was quantified by measuring the planar area of pseudopods with a computer-based video analysis system. When assessed 60 minutes following PAF treatment, the pseudopod area was significantly greater in PAF-treated cells than in control cells. In some calcitonin-treated osteoclasts, PAF induced pseudopod formation when applied focally using an extracellular micropipette, consistent with a direct action of PAF. We conclude that PAF directly induces pseudopod formation in calcitonin-inhibited osteoclasts, a morphologic response indicative of osteoclast activation.