bluuebunny/crossref_metadata_2025 huggingface dataset:
Choosing between predictors of fractures
Authors: Dr. Hui Siu L. , Slemenda Charles W. , Carey Mark A. , Johnston Conrad C.
Published: February 2010

The identification of those at highest risk of osteoporotic fractures is a clinical goal that requires appropriate statistical comparisons of potential predictors of fractures. This article provides a formal approach for comparing individual predictors (e.g., bone mass at one site vs bone mass at another), or sets of predictors (e.g., bone mass vs other risk factors), and contrasts newer methods, such as bootstrapping, to receiver-operating-characteristics (ROC) curves, which have been previously used. The advantages of the bootstrapping approach are illustrated using time-to-fracture data from a published study demonstrating the use of baseline bone mass measurements in the prediction of fractures in 521 subjects with variable lengths of follow-up, extending to 12.5 years. Bone mineral density (BMD) was shown to be significantly better than bone mineral content (BMC) in predicting fractures in free-living subjects, but not in retirement-community subjects. Bone mineral apparent density (BMAD) was also compared with BMC and BMD and shown not to improve fracture prediction in these subjects.
Effects of recombinant insulin-like growth factor-I and growth hormone on bone turnover in elderly women
Authors: Ghiron Laura J. , Thompson Janice L. , Holloway Leah , Hintz Raymond L. , Butterfield Gail E. , Hoffman Andrew R. , Marcus Robert
Published: February 2010

We evaluated the effects of recombinant insulin-like growth factor-I (IGF-I) and growth hormone (GH) on calciotropic hormones and bone turnover markers in 16 healthy elderly women 71.9 ± 1.3 years of age (mean ± SEM). Subjects consumed a fixed diet providing 1000 mg of calcium and 0.9 g/kg of protein for 10 days before starting baseline 24-h urine and blood collections. Specimens were collected for 6 consecutive days before initiating subcutaneous injections of GH (25 μg/kg/day, n = 5) and IGF-I at 60 μg/kg b.i.d. (high-dose, n = 5) or at 15 μg/kg b.i.d. (low-dose, n = 6) for 28 days. Resorption markers included urine hydroxyproline (OHP), total pyridinolines (PYD), and N-telopeptide; formation markers included osteocalcin, skeletal alkaline phosphatase (sALP), and type I procollagen carboxy-terminal extension peptide (CICP). For each subject, baseline daily turnover markers varied substantially (CV = 16–22%). With GH and high-dose IGF-I, resorption and formation markers increased progressively to maximum levels at day 21. For GH, the increase in day 21 PYD, N-telopeptide, osteocalcin, and CICP was 143, 111, 53, and 81%, respectively (p < 0.06–0.02). For high-dose IGF-I, these increases were 108, 81, 77, and 111% (p < 0.02–0.002). However, with low-dose IGF-I, no change was observed in resorption markers while osteocalcin and CICP increased progressively (day 21, % increases = 88 ± 51, 36 ± 14). Twenty-four hour urine collections during the last days of baseline and of study drug were taken as six 4 h aliquots. When deoxyPYD was measured on these samples in the low-dose IGF-I group, a significant increase was observed only on the 0800–1200 h aliquot. Serum phosphorus concentrations increased with GH (21.2 ± 3.3%) and high-dose IGF-I (8.8 ± 3.6%) by day 21 but actually decreased by day 28 (−9.7 ± 2.7, p < 0.02) with low-dose IGF-I. Urinary phosphorus excretion decreased with high-dose IGF-I only. Twenty-four hour calcium excretion increased with all treatments. These results indicate that both GH and high-dose IGF-I activate remodeling osteons. By contrast, low-dose IGF-I may directly increase osteoblastic function with only a minimal increase in bone resorption and may therefore provide a useful means to increase bone mass. The results also suggest some of the GH action on renal phosphorus handling represents a direct action of GH on the nephron which does not involve the intermediacy of IGF-I. Finally, even under controlled conditions bone turnover markers exhibit substantial daily variation so that a very large treatment effect will be required for these markers to have clinical utility.
The effects of recombinant human insulin-like growth factor (rhIGF)-1 and rhIGF-1/IGF binding protein-3 administration on rat osteopenia induced by ovariectomy with concomitant bilateral sciatic neurectomy
Authors: Narusawa Ken'Ichiro , Nakamura Toshitaka , Suzuki Katsumi , Matsuoka Yasushi , Lee Lyang-Ja , Tanaka Hiroyuki , Seino Yoshiki
Published: February 2010

We compared the effect of administration of recombinant human insulin-like growth factor-1 (rhIGF-1) alone or the rhIGF-1/IGF binding protein-3 (IGFBP-3) complex on osteopenia in rats. Female Sprague-Dawley rats (8 months old) underwent combined ovariectomy and bilateral sciatic neurectomy (OVX-NX) or sham operation only. After 2 months, the OVX-NX animals were injected subcutaneously with rhIGF-1 alone or with rhIGF-1/IGFBP-3 equimolar complex for 4 weeks. The IGF-1 contents and dose were 0.3 mg/kg of body weight (BW) three times/week, 3 mg/kg of BW once/week, or 3 mg/kg of BW three times/week. At the end of the experiment, the 4th and 5th lumbar vertebrae (L4, L5) and the proximal tibiae were removed after tetracycline labeling, and histomorphometrical analyses were performed on undecalcified sections using Villanueva's staining. The cancellous bone volume at L5 significantly increased by thickening of the trabecular width in rats treated with the complex. However, the increase in the values at the proximal tibia was not significant. The bone formation rates (BFR/BS) in the lumbar vertebrae of rats treated with the complex three times a week at doses of 0.3 mg/kg of BW and 3 mg/kg of BW were both significantly increased but the parameter increase was less marked with the dose of 3 mg/kg of BW once/week. The BFR/BS did not increase significantly in animals treated with IGF-1 alone. These findings clearly demonstrated that the effect of systemically administered rhIGF-1 on bone formation was markedly potentiated when combined with IGFBP-3 in estrogen deficiency combined with reduced activity. The action of IGF-1 was less potent on the bone in paralyzed limbs. The action of rhIGF-1/IGFBP-3 on trabecular bone appeared to depend not only on the dose but also on the frequency of administration and the parts of the skeleton in rats.
Parathyroid hormone-related protein is an autocrine modulator of rabbit proximal tubule cell growth
Authors: García-Ocaña Adolfo , de Miguel Fernando , Peñaranda Carlos , Albar Juan P. , Sarasa Jose L. , Esbrit Pedro
Published: February 2010

Parathyroid hormone-related protein (PTHrP), a likely mediator for humoral hypercalcemia of malignancy, is also synthesized in various normal tissues. In the kidney, PTHrP, mainly detected in proximal and distal tubules, has been shown to stimulate proliferation of rat mesangial cells in culture. Experiments were carried out to investigate the possible mitogenic effect of PTHrP in cultures of rabbit proximal tubule cells (PTC). Immunocytochemical analysis, using antihuman (h)PTHrP antibodies to (38–64) and (107–111) epitopes in the PTHrP molecule, showed strong cytoplasmic staining in PTC and in proximal tubule-like LLC-PK1 cells. PTC secreted immunoreactive PTHrP (54.8 ± 7.0 fmol/106 cells) into the culture medium. Human PTHrP(1–141) stimulated proliferation in subconfluent cultures of these cells dose-dependently. This effect was similar to that induced by [Tyr34]hPTHrP(1–34) amide (hPTHrP[1–34]), hPTHrP(1–86), and bovine (b)PTH(1–34), while hPTHrP(38–64) amide, hPTHrP(107–111) amide, and hPTHrP(107–139) amide were ineffective. Addition of anti-hPTHrP neutralizing antibodies to (1–34), (38–64), and (107–111) epitopes of PTHrP decreased PTC growth. The mitogenic effect of these agonists was abolished in confluent PTC. In contrast, [Nle8,18, Tyr34]bPTH(3–34) amide (bPTH[3–34]) increased DNA synthesis in either subconfluent or confluent PTC. In LLC-PK1 cells, which also secreted PTHrP and are devoid of PTH receptors, none of these peptides affected proliferation. Forskolin (10 μM) or H-8 (2 μM), a protein kinase A inhibitor, did not affect basal or hPTHrP(1–34)-stimulated DNA synthesis, respectively, in subconfluent PTC. On the other hand, 10 nM staurosporine and 100 nM calphostin C, protein kinase C (PKC) inhibitors, blunted the effects of hPTHrP(1–34) or bPTH(3–34) on DNA synthesis in these cells. These studies suggest that PTHrP may function as an autocrine factor in the regulation of proximal tubule cell growth by a PKC-mediated mechanism.
Circadian variation in urinary excretion of bone collagen cross-links
Authors: Bollen Anne-Marie , Martin Michael D. , Leroux Brian G. , Eyre David R.
Published: February 2010

Bone resorption can be evaluated by measuring the urinary excretion of collagen type I cross-linked N telopeptides (NTx). Since it is difficult to obtain (and verify) 24 h urine collections from patients, untimed spot urines are more practical. Such measurements, however, need correction for urine dilution and potentially may vary with collection time since a circadian rhythm in bone metabolism has been reported. This study examined cross-link excretion in urine voids serially collected during a 24 h period from subjects living their normal daily routine (as opposed to a controlled hospital setting). This mimics the situation for walk-in patients visiting a clinician and providing a spot urine. A total of 35 dentists (20 males, 15 females) collected all urine voids separately over a 24 h period. Urines were analyzed for creatinine and NTx. The effects of time of day on the excretion rates of these metabolites (in nmol/h) and on the cross-link:creatinine ratio were assessed. A circadian rhythm was evident in the excretion rate of creatinine with a peak in the late afternoon (18% higher than the 24 h mean, p = 0.0004). The NTx excretion rate peaked in the morning (9% higher than the 24 h mean) but this latter rhythm was not statistically significant (p = 0.31). The NTx:creatinine ratio fell during the day from a high (122% of the 24 h mean) in the early morning to a low in the early evening. This rhythm in the NTx:creatinine ratio in untimed spot urines was statistically significant (p < 0.0001). In conclusion, the NTx:creatinine ratio in spot urines from adult outpatient subjects showed a significant circadian rhythm. Variations in creatinine excretion were the primary cause. Time of day should, therefore, be taken into account when comparing test results of spot urines with normal ranges or with other samples from the same subject.
Regulation of gap junction intercellular Communication by pH in MC3T3-E1 osteoblastic cells
Authors: Yamaguchi Dean T. , Huang Jason T. , Ma Defang
Published: February 2010

Gap junction intercellular communication (GJIC) may be related to coordinating the function of osteoblasts during bone mineralization. Since an alkaline pH supports mineral deposition while an acidic pH promotes mineral dissolution, it was investigated whether GJIC is altered by changes in extracellular pH (pHo) Functional GJIC was assessed by fluorescent dye transfer after microinjection, and connexin protein abundance was examined by immunoprecipitation and immunoblotting in MC3T3-E1 cells, a model of osteoblast-like cells. The percent of cells coupled by GJIC was found to be 40.7% (24 of 59 injected cells) at pH 6.9, 72.2% (26 of 36) at pH 7.2, and 92.8% (26 of 28) at pH 7.6. A decrease in GJIC was detectable by 30–60 minutes of exposure to a pHo of 6.9. Decreased gap junction communication was also found in cells after 3, 8, and 24 h of incubation in a bicarbonate-CO2 system at an ambient pH of 6.9. Connexin protein abundance experiments showed that at after exposure to a pH of 6.9 for 2.75 h, the specific band(s) at 41–43 kD were fainter compared with these same band(s) at pH 7.2 and 7.6. There was no significant difference in band densities at pH 7.2 and 7.6. Determination of intracellular pH (pHi) showed that it was similar to pHo after 2.75 h of incubation at each ambient pH. When pHi was clamped at 6.9 or 7.2, there was a time-dependent decrease in the gap junction coupling frequency at a pHi of 6.9 when pHo was 7.2. Steady-state mRNA levels were decreased at pHo 6.9 but were unchanged at either pHo 7.2 or 7.6. Our conclusions are that (1) longer incubations (≥2.75 h) at low pHo decrease GJIC which in part may be due to a decrease in connexin protein abundance perhaps as a result of a decrease in connexin steady-state mRNA expression; (2) GJIC inhibition or augmentation found at low and high pHo, respectively, suggests that gating of the GJ channel by pH may also occur; (3) pHo-induced alterations in GJIC in the MC3T3-E1 osteoblastic model are related to concomitant changes in pHi.
Predictors of hip fractures in elderly men
Authors: Poór Gyula , Atkinson Elizabeth J. , O'Fallon W. Michael , Melton L. Joseph
Published: February 2010

To assess the influence on the risk of hip fractures in men of medical conditions associated with secondary osteoporosis or with an increased likelihood of falling, we conducted a population-based nested case-control study among the 232 Rochester, Minnesota, men with an initial hip fracture due to moderate trauma in 1965–1989 and an equal number of age-matched control men from the general population. Information on selected medical and surgical conditions and certain behavioral risk factors prior to fracture (or comparable index date for controls) was obtained from inpatient and outpatient medical records in the community that averaged over 36 years in duration. After adjusting for age, obesity, and inactivity, disorders linked with secondary osteoporosis were associated with a 2-fold increase in the risk of hip fracture in men (odds ratio [OR] 2.3; 95% confidence interval [CI] 1.3–4.3), while conditions linked with an increased risk of falling were associated with almost a 7-fold increase in risk (OR 6.9; 95% CI 3.3–14.8). These factors together appeared to account for about 72% of the hip fractures in men. Increased attention must be paid to these conditions which, in aggregate, are very common in elderly men and lead to a substantial increase in the risk of hip fracture with its devastating sequelae of death, disability, and cost.
Measurement of femoral geometry in type I and type II osteoporosis: Differences in hip axis length consistent with heterogeneity in the pathogenesis of osteoporotic fractures
Authors: Boonen Steven , Koutri Rosetta , Dequeker Jan , Aerssens Jeroen , Lowet Geert , Nijs Jos , Verbeke Geert , Lesaffre Emmanuel , Geusens Piet
Published: February 2010

The epidemiologic patterns of vertebral and femoral fractures are sufficiently different to suggest that they represent distinct disorders (type I versus type II osteoporosis) although osteopenia is common in both. To determine whether differences in femoral geometry, one of the main determinants of bone quality, might contribute to the heterogeneity in osteoporotic fractures, we obtained dual energy X-ray absorptiometry scans on 210 women age 60 or older, including 105 type 1 fracture cases, 30 type II patients, and 75 controls. Hip axis length, measured on the scan printout, was significantly increased (p < 0.01) in hip fracture patients compared with women with postmenopausal osteoporosis, whereas femoral neck density (BMD) was equal in both groups. The best discrimination between both fracture types was obtained by a logistic regression model based on age and axis length. Adding BMD to the model did not improve the discriminative power (p = 0.67). These data provide further evidence that geometric characteristics may be implicated in hip fracture risk. Furthermore, these findings suggest that an increase in hip axis length may predispose osteopenic subjects to a femoral localization of fragility fractures, consistent with the postulated heterogeneity in the pathogenesis of osteoporotic fractures.
Equivalent deficits in bone mass of the vertebral body and posterior processes in women with vertebral fractures: Implications regarding the pathogenesis of spinal osteoporosis
Authors: Prof. Seeman Ego , Formica Carmelo , Mosekilde Lis
Published: February 2010

Reduced bone mass of the spine in women with vertebral fractures is attributed to excessive trabecular bone loss from the vertebral body. However, the measurement obtained by posteroanterior (PA) scanning includes the posterior processes and the vertebral body, each comprising about 50% of the total vertebral mass. Thus, the deficit in bone mass by PA scanning may be due to deficits in one or both of these structures. We asked two questions: (1) In healthy women, is the age-related diminution in bone mass of the vertebral body greater than the diminution at the posterior processes? (2) In women with vertebral fractures, is the deficit in bone mass at the vertebral body, the fracture site in spinal osteoporosis, greater than at the posterior processes? Bone mass of the posterior processes and vertebral body of the third lumbar vertebra was measured by lateral scanning using dual-energy X-ray absorptiometry (DXA). Compared with 27 premenopausal women, deficits in 27 postmenopausal women at the posterior processes and vertebral body, respectively, were 35.9 ± 3.7 and 25.2 ± 4.1% (p < 0.05); t score, –1.5 ± 0.2 and –1.1 ± 0.2 SD (p = 0.09). Compared with the postmenopausal (age-matched) women, deficits in 21 women with vertebral fractures at the posterior processes and vertebral body, respectively, were 22.6 ± 4.9 and 24.5 ± 8.3% (p = NS); Z score, –0.8 ± 0.2 and –0.8 ± 0.3 (p = NS). In vivo the bone mass of the vertebral body as a percentage of the whole vertebra was 45.7 ± 0.1 in premenopausal women, 48.9 ± 1.9 in postmenopausal women, 51.5 ± 1.1 in women with low bone mass but no fractures, 52.7 ± 2.4 in women with vertebral fractures, and 51.9 ± 2.5% in vitro, based on autopsy specimens from 19 postmenopausal women aged 65–95 years. The lower spinal bone density measured using PA scanning in women with spine fractures may not be due to excessive or disproportionate trabecular bone loss from the vertebral body because comparable deficits are found at the posterior processes. Whether these deficits are due to reduced peak bone mass, trabecular bone loss, cortical bone loss, or varying combinations of these mechanisms remains to be established.
Segregation analysis and variance components analysis of bone mineral density in healthy families
Authors: Guéguen René , Jouanny Pierre , Guillemin Francis , Kuntz Claude , Pourel Jacques , Siest Gérard
Published: February 2010

Bone mineral density (BMD) was measured in 1992–93 in 129 nuclear families, including 258 parents and 183 children, and was analyzed for familial resemblance factors. BMD measurements were adjusted on weight and age. Segregation analysis rejected the monogenic hypothesis and exhibited a strong polygenic component. Variance components analysis was then used to estimate the parameters of a multivariate normal model including an additive polygenic component, a common environmental factor, and a residual specific to each individual. The genetic component was independent of sex and age. The common environmental factor was not significant. The variance of the residual specific factor appeared to be a quadratic function of age, reaching its minimum value at 26.4 years. Consequently, the maximum value for heritability (ratio of genetic variance to total variance) is observed at this age (h2 = 0.84). According to this model, the correlation between two relatives is a function of the ages of each individual in the pair.
Elevated progesterone during pseudopregnancy may prevent bone loss associated with low estrogen
Authors: Bowman Beth M. , Miller Scott C.
Published: February 2010

There is increasing evidence for a role of endogenous progesterone in mineral and skeletal metabolism. The purpose of this study was to compare skeletal changes that occur during a condition of high endogenous progesterone but low estrogen (pseudopregnancy) with a condition of low endogenous progesterone and low estrogen (ovariectomy). Pseudopregnancy was selected over pregnancy to eliminate placental factors that may influence mineral metabolism. Rats were ovariectomized (OVX) or pseudopregnancy initiated, and bones were collected 13 days later. In some animals, blood was collected by indwelling catheters for determination of progesterone and estrogen levels. At mid-pseudopregnancy, there were substantial elevations in progesterone but estrogen was below the level of detection. Progesterone and estrogen were below the level of detection in the OVX rats. Longitudinal growth rates were increased compared with the normal cycling rats in both the pseudopregnant and OVX groups, indicative of decreased estrogen levels, but they were greatest in the OVX rats. Cancellous bone mass was maintained in the pseudopregnant rats compared with normal cycling rats but significantly reduced in the OVX animals. As expected, increased bone formation and turnover rates were observed in the OVX animals although some indices of bone formation were also increased in the pseudopregnant animals. Osteoclasts were significantly increased in the OVX but not the pseudopregnant animals compared with normal cycling rats. Increased periosteal bone formation indices are known to occur following OVX, but the greatest periosteal formation rates were observed in the pseudopregnant animals. While possible roles for some other endocrine agents cannot be excluded at this time, the data from the present study suggest that endogenous progesterone may have a role in the maintenance of bone mass perhaps by decreasing bone resorption while maintaining or increasing bone formation during physiological periods of low estrogen such as occurs during early pregnancy.
Ontogeny of the 1,25-dihydroxyvitamin D3 receptor in fetal rat bone
Authors: Johnson Julie A. , Grande Joseph P. , Roche Patrick C. , Dr. Kumar Rajiv
Published: February 2010

To gain insights into 1,25-dihydroxyvitamin D3 receptor (VDR) function during fetal bone development, we examined fetal rat tissues from gestational days 13–21 for the presence and distribution of VDR using immuno-histochemistry. Prior to ossification, VDR epitopes were observed in the mesenchyme condensing to form skeletal tissues, on day 13 in the developing vertebral column and limbs, and on day 17 of gestation in developing calvaria. Immunostaining for VDR was seen in proliferating and hypertrophic chondrocytes and in osteoblasts of limb buds and the vertebral column by day 17 of gestation. In calvaria, VDR epitopes were observed in osteoblasts by gestational day 19. VDR immunostaining was also evident in the skin of fetal limbs at all gestational ages examined. We show for the first time that the VDR appears very early in the developing fetal rat skeleton, suggesting that the VDR, in concert with its ligand, 1,25-dihydroxyvitamin D3, may play a role in the differentiation of mesenchymal precursors into bone tissue.
Expression of amelogenin mRNA sequences during development of rat molars
Authors: Wurtz Tilmann , Lundmark Carin , Christersson Cecilia , Bawden James W. , Slaby Ivan , Hammarström Lars
Published: February 2010

The expression of amelogenin mRNA in growing rat molars was studied. Northern blotting and the analysis of cDNA isolates revealed two predominant variants. One group of cDNA inserts contained sequences of a long mRNA version and the other group contained mRNA sequences of the shorter leucin-rich amelogenin polypeptide (LRAP). The LRAP group was deficient in an internal stretch which coded for a peptide with a high potential for beta turns. Northern blot experiments showed that most amelogenin RNA in rat teeth was represented by two bands of 1.1 and 0.8 kb. Two oligonucleotide probes were designed that were specific for the long version and for the LRAP variant. The probes were used for in situ hybridization experiments on sections of developing maxillar teeth of rats between day 2 and day 15 after birth. Both RNA species were accumulated concomitantly and exclusively in cells of the inner enamel epithelium. Expression was first observed at the mesial cusp sides and finally involved the whole ameloblast layer except for the cells adjacent to the enamel-free region at the tip of the cusps. The early amelogenin RNA expression occurred adjacent to the initial deposition of the dentin matrix. Low amounts of amelogenin RNA persisted after the differentiation of ameloblasts into the maturative stage. The sequence of events was similar in all three molars.
Expression of the nucleoside triphosphate pyrophosphohydrolase PC-1 is induced by basic fibroblast growth factor (bFGF) and modulated by activation of the protein kinase A and C pathways in osteoblast-like osteosarcoma cells
Authors: Solan Joell L. , Deftos Leonard J. , Coding James W. , Terkeltaub Robert A.
Published: February 2010

The closely related cytokines bFGF and aFGF regulate the function of bone cells and mineralization. Osteoblasts express PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH)/nucleotide phosphodiesterase I activity. bFGF and aFGF (10 ng/ml) up-regulated NTPPPH in human SaOS-2 and U2OS osteosarcoma cells, which express osteoblast-like features in culture. The induction was selective as alkaline phosphatase activity was down-regulated and specific as insulin-like growth factor-1 (IGF-1) and interleukin-1β (IL-1β) were not active. Furthermore, IL-β but not IGF-1 inhibited bFGF-induced up-regulation of NTPPPH. The induced NTPPPH remained predominantly associated with cells. bFGF can induce signaling through pathways including protein kinase A (PKA) and protein kinase C (PKC)-mediated transduction. An activator of the PKA pathway (8-bromo cyclic adenosine monophosphate [cAMP]) induced NTPPPH. Furthermore, pretreatment with the PKC activator phorbol myristate acetate (PMA) (80 nM) markedly increased subsequent NTPPPH induction by both bFGF and cAMP. The PMA effect was associated with morphologic changes characterized by long, thin intercellular extensions. PKC desensitization also potentially contributed to this effect because the PKC inhibitors staurospo-rine and H-7 enhanced bFGF-induced and cAMP-induced NTPPPH expression in the absence of morphologic changes. We observed that bFGF induced expression of PC-1, a member of the NTPPPH gene family. The majority of NTPPPH activity was depleted by immunoadsorption using a monoclonal antibody to native human PC-1. bFGF- and aFGF-induced production of PC-1/NTPPPH in osteoblastoid cells may contribute to the effects of FGFs on bone metabolism.
Quantification of the collagenolytic activity of isolated osteoclasts by enzyme-linked immunosorbent assay
Authors: Foged Niels Taekker , Delaisse Jean-Marie , Hou Peng , Lou Henriette , Sato Takuya , Winding Bent , Bonde Martin
Published: February 2010

Difficulties in the geometrical definition and measurement of resorption pits is a major problem for the quantitative analysis of bone resorption by isolated osteoclasts cultured on bone or dentin substrates. In this study we developed an enzyme-linked immunosorbent assay (ELISA) for quantification of bone resorption in vitro, which specifically quantifies type I collagen fragments released into the culture medium by the resorptive action of bone cells cultured on slices of bone or dentin. A consistently high correlation between the formation of resorption pits and the release of antigenic collagen fragments was observed for isolated rabbit osteoclasts seeded at various densities and cultured for various periods on bovine, elephant, and human substrates. In a further support of the osteoclastic nature of the collagenolytic effects, a high consistency between pit formation and collagenolysis was also observed when the rabbit bone cells were cultured in the presence of very differently acting but typical inhibitors of pit formation, i.e., the carbonic anhydrase inhibitor acetazolamide, the cysteine proteinase inhibitor epoxysuccinyl-L-leucylamido-(4-guanodino)butane (E-64), the phosphatidyl-inositol 3-kinase inhibitor wortmannin, and the bisphosphonate ibandronate (BM 21.0955). In conclusion, the ELISA represents a simple, precise, and objective way to dynamically monitor bone resorption in vitro through quantification of the collagenolytic activity of isolated osteoclasts.
Prediction of perimenopausal fractures by bone mineral density and other risk factors
Authors: Torgerson D.J. , Campbell M.K. , Thomas R.E. , Reid D.M.
Published: February 2010

In a prospective population-based cohort study, we assessed whether bone mineral density (BMD) measurements of perimenopausal women and other risk factors for osteoporosis are predictive of subsequent fracture. Women aged 47-51 years chosen randomly from a population register who underwent a bone density measurement 2 years previously were followed up by questionnaire to assess the 2-year incidence of any self-reported fractures. We found that 44 women, out of 1857 who completed the questionnaire, sustained at least one fracture within a 2-year follow-up period. After adjustment for covariates, the odds ratio of sustaining a fracture was 1.6 (95% confidence interval [CI] 1.16-2.34) for every standard deviation reduction in BMD at the spine, for women with a prior history of fracture the odds ratio of a subsequent fracture was approximately 2 (95% CI 1.31–3.03), a family history of hip fracture (maternal grandmother) carried an odds ratio of 3.7 (95% CI 1.55-8.85), while being postmeno-pausal or having a hysterectomy resulted in an odds ratio of 1.98 (1.02-3.56). This study has shown that BMD measurements at the hip and spine and other risk factors predict any nonhip and nonspine perimenopausal fractures. Further follow-up is required to assess the predictive performance of BMD measurements and other risk factors for hip and spine fractures.
Nitric oxide and bone
Authors: Dr. Evans Deborah M. , Ralston Stuart H.
Published: February 2010

Nitric oxide (NO), a mediator of cardiovascular homeostasis, neurotransmission, and immune function, has recently been found to have important effects in bone. Both constitutive and inducible forms of NO synthase are expressed by bone-derived cells, and cytokines such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and interferon gamma (IFN-γ), are potent stimulators of NO production. When combined with other cytokines, IFN-γ markedly induces NO production, which suppresses osteoclast formation and activity of mature osteoclasts. This “superinduction” of NO is largely responsible for the selective inhibitory effect of IFN-γ on cytokine-induced bone resorption. High concentrations of NO are also inhibitory for cells of the osteoblast lineage, and NO production appears to be partly responsible for the inhibitory effects of cytokines on osteoblast proliferation. At lower concentrations, however, NO has different effects. Moderate induction of NO potentiates bone resorption, and the constitutive production of NO at low concentrations promotes the proliferation of osteoblast-like cells and modulates osteoblast function. NO therefore appears to be an important regulatory molecule in bone with effects on cells of the osteoblast and osteoclast lineage and represents one of the molecules produced by osteoblasts which directly regulate osteoclastic activity. Stimulation of NO production in bone by proinflammatory cytokines raises the possibility that NO may be involved as a mediator of bone disease in conditions associated with cytokine activation, such as rheumatoid arthritis, tumor associated osteolysis, and postmenopausal osteoporosis.
Isolation and characterization of osteoblast precursor cells from human bone marrow
Authors: Rickard David J. , Kassem Moustapha , Hefferan Theresa E. , Sarkar Gobinda , Spelsberg Thomas C. , Riggs B. Lawrence
Published: February 2010

Osteoblasts are derived from precursor cells present in low frequency in the stromal element of bone marrow. Because of the lack of a practical procedure to isolate osteoblast precursors from early cultures of plastic adherent cells from bone marrow, previous studies of marrow stromal cells have been made in confluent cultures of bone marrow when the osteoblast (OB) precursors are already differentiated. Also these studies utilized cultures containing mixed populations of cells including hematopoietic cells. Thus we have employed a negative immunoselection procedure to remove contaminating hematopoietic cells and to isolate nearly homogeneous populations of early human stromal cells derived from the plastic-adherent mononuclear marrow cells cultured in the presence of serum. By reverse transcriptase polymerase chain reaction (RT-PCR) analysis for mRNA, and by immunocytochemical study for protein, we studied the sequential expression in culture of multiple markers of the osteoblast phenotype-alkaline phosphatase, osteopontin, parathyroid hormone receptor, types I and III procollagen, and osteocalcin-as well as lipoprotein lipase (LPL), a marker of the adipocyte phenotype. At an early stage of culture (7-9 days), human OB precursors formed colonies of variable sizes that expressed low levels of mRNA and protein concentrations of OB markers, and their concentration increased on growth to a confluent monolayer (approximately 14 days). LPL mRNA was expressed at high levels in the colony stage, and its level decreased upon confluency, suggesting a loss of potential for commitment to the adipocyte lineage. Interestingly, treatment with dexamethasone at 10−8 M increased the expression for some of the osteoblast markers and for the LPL gene and was required for the deposition of mineralized matrix and for the formation of adipocytes containing cytoplasmic lipid droplets in confluent cultures. Cloned single early colonies were able to coexpress the osteoblast and adipocyte markers (as assessed by RT-PCR). Thus these immunoselected marrow stromal cells have the characteristics of authentic human osteoblast precursor cells which also are capable of differentiating into adipocytes.
Effects of a synthetic vitamin D analog, ED-71, on bone dynamics and strength in cancellous and cortical bone in prednisolone-treated rats
Authors: Tanaka Yuzo , Dr. Nakamura Toshitaka , Nishida Satoshi , Suzuki Katsumi , Takeda Satoshi , Sato Katsuhiko , Nishii Yasuho
Published: February 2010

To determine the action of corticosteroid on bone metabolism and assess the effects of a synthetic vitamin D analog, ED-71, on them, 56 SD rats, 8 weeks of age, were assigned to seven groups of eight animals each. Group 1 was the basal control. Group 2 was the nontreated control. Groups 3–7 were given prednisolone at 30 mg/kg of body weight (BW) twice a week and concomitantly administered ED-71 with respective doses of 0, 0.0125, 0.025, 0.05, and 0.1 μg/kg of BW for 12 weeks. In group 3, urinary calcium (U-Ca) and deoxypyridinoline (U-Dpy) were significantly increased compared with group 2. In groups 4–7, U-Ca values were increased but U-Dpy values were dose-dependently decreased. Age-dependent increases in the parameter values of BMD, compressive strength, trabecular bone volume (BV/TV), and trabecular thickness (Tb.Th) of the lumbar body were significantly suppressed in group 3 but dose-dependently increased in groups 4–7, and the values of group 7 exceeded those of group 2. The parameters of bone mineral density (BMD) and the bending strength of the femur in groups 4–7 were larger than the values in group 3 but did not reach the levels of group 2. The trabecular bone formation rate (BFR/BS) of the lumbar body measured by calcein labeling in group 3 was reduced when compared with group 2, but the values were not further decreased in groups 4–7. The perimeter ratios of double labels over single labels (dLS/sLS) greatly decreased by prednisolone, were dose-dependently increased to the level of the normal control by ED-71. Double-labeled perimeters and the dLS/sLS ratios were also increased in the periosteal envelope of the midfemur. These findings clearly demonstrate that prednisolone administration affects the age-related changes in bone metabolism, and ED-71 administration counteracts the effects by increasing intestinal calcium absorption, reducing bone resorption, and enhancing mineralization. The action of ED-71, however, seems to be less potent in the cortical bone.
TGF-β and basement membrane matrigel stimulate the chondrogenic phenotype in osteoblastic cells derived from fetal rat calvaria
Authors: Basic Nikolina , Basic Vanja , Bulic Kresimir , Grgic Marko , Kleinman Hynda K. , Luyten Frank P. , Vukicevic Slobodan
Published: February 2010

Primary cultures of fetal rat calvarial cells contain a spectrum of osteogenic phenotypes including undifferentiated mesenchymal cells, osteoprogenitor cells, and osteoblasts. We recently demonstrated that rat calvarial osteoblast-like cells grown on basement membrane undergo profound morphological changes resembling a canalicular network in bone. In the present study, we examined the effect of reconstituted basement membrane Matrigel on chondroblastic versus osteoblastic differentiation of different cell subpopulations obtained by five consecutive enzymatic digestions of rat calvarial cell populations. We found that the appearance of canalicular cell processes decreased with the later digests. When cells from the fourth and fifth digest were grown on top of Matrigel for 7 days, the majority of the cell aggregates displayed chondrocytic characteristics but none of the cells became hypertrophic. When individual chondroblastic cell aggregates were subsequently transferred from Matrigel to plastic, they started expressing types I and X collagens, alkaline phosphatase, and osteocalcin. Within the next 7 days (days 8–14 of the experiment), the majority of cells increased in size, and at day 17 on plastic (day 24 of the experiment) mineralized bone nodules formed. The chondroblastic differentiation of calvarial cells grown on Matrigel could be inhibited by a specific transforming growth factor-β1 (TGF-β1) but not by a TGF-β2 antibody. Addition of recombinant TGF-β1 to similar cultures promoted the appearance of chondroblastic cell aggregates. The cartilage phenotype could not, on the contrary, be promoted by growing the cells on other extracellular matrices such as a collagen I gel. We suggest that TGF-β1 in concert with the basement membrane extracellular matrix induces chondroblastic differentiation of rat calvarial osteoprogenitor cells.
Involvement of prostaglandin endoperoxide H synthase-2 in osteoclast-like cell formation induced by interleukin-1β
Authors: Sato Takahiro , Dr. Morita Ikuo , Sakaguchi Kouji , Nakahama Ken-Ichi , Smith William L. , Dewitt David L. , Murota Sei-Itsu
Published: February 2010

Interleukin-1β (IL-1β) stimulates osteoclast-like cell formation via prostaglandin E2 (PGE2) production. However, the regulatory mechanism for the production of PGE2 in bone cells is still unclear. Recently, it has been shown that two prostaglandin endoperoxide H synthase (PGHS) isozymes exist, termed PGSH-1 and PGHS-2. We report here that IL-1β induces PGE2 production in bone marrow culture induced by a PGHS-2-dependent mechanism. IL-1β stimulated the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC) and the production of PGE2 in mouse bone marrow cultures. The dose response curves for the indomethacin inhibition of TRAP-positive MNC formation and PGE2 production were nearly identical. Cycloheximide (CHX) suppressed IL-1β-induced PGE2 production, suggesting that the production of PGE2 induced by IL-1β required de novo protein synthesis. Northern blot analysis determined that IL-1β induced PGHS-2 expression by 30 minutes and mRNA levels were maximal by 1–2 h. Cycloheximide potentiated the accumulation of PGHS-2 mRNA linearly up to 8 h. Dexamethasone, an inhibitor of the induction of PGHS-2, inhibited IL-1β-induced PGHS-2 mRNA expression and also suppressed IL-1β-stimulated formation of TRAP-positive MNC. Furthermore NS-398, as a selective PGHS-2 inhibitor, completely inhibited IL-1β-induced TRAP-positive MNC formation. Moreover, IL-1β-induced PGHS-2 mRNA expression and formation of TRAP-positive MNC were inhibited by calphostin C, a selective inhibitor of protein kinase C (PKC). These results indicate that IL-lβ-induced formation of osteoclast-like cells requires PKC activation, induction of PGHS-2, and subsequent prostaglandin synthesis by this enzyme.
Effect of physical activity as a caddie on ultrasound measurements of the os calcis: A cross-sectional comparison
Authors: Dr. Hoshino Hironobu , Kushida Kazuhiro , Yamazaki Kaoru , Takahashi Masaaki , Ogihara Hiroaki , Naitoh Kenichi , Toyoyama Okimitsu , Doi Syun , Tamai Hideki , Inoue Tetsuo
Published: February 2010

This cross-sectional study investigated the effect of long-term activity as a caddie on ultrasonic properties of the os calcis. We measured 74 healthy women, age 20–59 years, who worked at a golf course as caddies. An age-matched control group of 433 healthy women, who were office workers or housewives, also were recruited for comparison. The ultrasound measurements were performed with an Achilles ultrasound densitometer. The quadriceps muscle strength and the hand grip strength were measured in a perimenopausal subgroup (45-59 years) of the caddies and a subgroup of controls matched for age, height, weight, and body mass index. Urinary pyridinoline and deoxypyridinoline were also measured in these perimenopausal subgroups. Caddies had significantly higher ultrasound values than controls in the 40–49 (stiffness index, 101.6 ± 12.9% versus 87.9 ± 11.9%; p < 0.0001) and 50–59 (stiffness index, 90.5 ± 11.6% versus 77.2 ± 11.6%; p < 0.0001) age-stratified groups. Quadriceps strength and grip strength were significantly higher in caddies than those in controls. In postmenopausal caddies, all ultrasound values were significantly higher than for controls. In caddies there were not significant decreases of any ultrasound values with postmenopausal age. Even for the subgroup within 3 years of menopause there were significant differences between caddies and controls (p < 0.01). There were no significant increases of pyridinoline and deoxypyridinoline after menopause in the caddies. We demonstrated that the caddies had higher ultrasound properties of the os calcis and lower bone resorption after menopause compared with controls.
Temporal expression of the anabolic action of PTH in cancellous bone of ovariectomized rats
Authors: Meng X.W. , Liang X.G. , Birchman R. , Wu D.D. , Dempster D.W. , Lindsay R. , Shen V.
Published: February 2010

When administered intermittently, parathyroid hormone (PTH) is a potent anabolic agent in both human and animal bone. To improve our understanding of this anabolic effect, we have examined the time course of PTH action in an established animal model of estrogen deficiency-induced bone loss: the ovariectomized rat. Animals were ovariectomized (Ovx) and allowed to lose bone for 6 weeks. A dose of 20 μg/kg/d of rat PTH (1–34) was administered s.c., 6 days each week for periods of 1, 2, 3, 4, 6 and 8 weeks. Animals were sacrificed for evaluation of skeletal histomorphometry of the proximal tibia and mechanical strength of the cancellous bone in the marrow cavity of the distal femur. Cancellous bone volume (Cn-BV/TV) increased gradually over 8 weeks of treatment (16.8 ± 1.6 to 24.1 ± 2.7%) as did the bone formation rate (0.308 ± 0.054 to 1.659 ± 0.293 μm3/μm2/d), as determined by an increase in both total mineralization surface (15.5 ± 2.1 to 42.7 ± 5.0%) and mineral apposition rate (1.88 ± 0.20 to 3.55 ± 0.39 μm/d). The largest increments in these variables reflecting bone formation occurred over the first week of treatment. This bone formation was accompanied by an increase in trabecular thickness (Tb.Th) (55.3 ± 3.4 to 80.5 ± 5.0 μm) without a corresponding increment in trabecular number (Tb.N) (3.65 ± 0.17 to 3.55 ± 0.26). Extensive tetracycline labels were visualized on the surface of trabecular rod-like and plate-like structures. A small transient, though not statistically significant, increase occurred in both eroded surface and urinary pyridinoline concentration immediately after the onset of PTH administration. Osteocalcin showed a small decrement in the first two weeks after PTH administration, but the levels were elevated when compared with the Ovx control in later weeks. Mechanical strength of the cancellous bone also increased significantly with PTH treatment (20.5 ± 2.4 to 46.1 ± 10.0 Newtons). Our results showed that: 1) intermittent PTH treatment of Ovx rats elicited an immediate increase of bone formation activity by the existing osteoblasts, 2) the increase of Cn-BV/TV after PTH administration resulted primarily from an increase in Tb.Th, and 3) improved mechanical strength after PTH treatment can be achieved by increases in Tb.Th without an increase in Tb.N.
Effects of synthetic peptido-leukotrienes on bone resorption in vitro
Authors: Garcia C. , Qiao M. , Chen D. , Kirchen M. , Gallwitz W. , Mundy G.R. , Bonewald L.F.
Published: February 2010

Peptido-leukotrienes are short-lived organic molecules known to have potent biological effects as mediators of inflammation, hypersensitivity and respiratory disorders. However, little is known concerning their effects on bone cells. We have shown previously that stromal cells isolated from a human giant cell tumor secrete 5-HETE (5-hydroxyeicosatetraenoic acid) and the peptido-leukotrienes, also known as the cysteinyl leukotrienes LTC4, LTD4, and LTE4. These eicosanoids were shown to stimulate the multinucleated giant cells obtained from these tumors to form resorption lacunae on sperm whale dentine. Here, we show that the peptido-leukotrienes also stimulate isolated avian osteoclast-like cells to form resorption lacunae and to increase their content of tartrateresistant acid phosphatase. LTD4 increased 45Ca release from murine calvarial bone organ cultures, but not from fetal rat long bone cultures. Isolated avian osteoclast-like cells were chosen to perform receptor binding studies, as this population is the most homogenous source of osteoclasts available. After the precursors had fused to form multinucleated cells, receptor binding assays were performed. Scatchard analysis of saturation binding data showed a single class of binding sites, with a dissociation constant (Kd) of 0.53 nM and a receptor density of 5,200 receptors per cell. Competition binding studies showed receptor specificity using a specific LTD4 receptor antagonist ZM 198,615. These data show that the peptido-leukotrienes activate highly enriched populations of isolated avian osteoclast-like cells, and also that specific LTD4 receptors are present in this cell population.