bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental expression of bone sialoprotein mRNA in rat mineralized connective tissues
Authors: Chen Jinkun , Shapiro Howard S. , Sodek Jaro
Published: February 2010

Bone sialoprotein (BSP) is a phosphorylated and sulfated glycoprotein that is a major noncollagenous protein of bone and other mineralizing connective tissues. BSP is characterized by the presence of several polyglutamic acid segments and an RGD motif that mediates cell attachment through a vitronectin-like receptor. Although the precise function of BSP is unknown, the expression of BSP in conjunction with bone formation in vitro indicates a role for this protein in the biomineralization of connective tissues. In this study we used Northern hybridization and in situ hybridization to determine the tissue-specific and developmental expression of BSP during embryogenesis and growth of rat tissues. Analysis of tissues obtained from 13, 17, and 21 day fetuses, and from 4-, 14-, and 100-day-old animals indicates that BSP mRNA expression is restricted to cells actively forming the mineralizing tissues of bone, dentin and cementum. BSP mRNA transcripts were first evident in fully differentiated osteoblasts of 17 day fetal tissues at sites of de novo intramembranous and endochondral bone formation, with maximal expression observed at 21 days of gestation. Thereafter, BSP mRNA levels decreased markedly, and in adult bone hybridization was detected only in the primary spongiosa of long bones. In comparison, mRNAs for osteopontin (OPN), alkaline phosphatase (ALP), and osteocalcin (OC) peaked at 4–14 days postpartum before declining. In the tibiae, Northern hybridization revealed a second peak of mRNA for BSP, ALP, and OPN at 14 days, reflecting an increased osteogenic activity due to the formation of the secondary centers of ossification in the epiphyseal cartilage. In situ hybridization also revealed BSP mRNA in hypertrophic chondrocytes at sites of bone formation, in odontoblasts of the incisor during dentinogenesis, and in cementoblasts during cementogenesis. In view of the restricted distribution and temporal changes in the expression of BSP mRNA that we observed together with the chemical properties of BSP, we believe that this protein has a specific role in mediating the initial stages of connective tissue mineralization.
Perspective how many women have osteoporosis?
Authors: Dr. Melton L. Joseph , Chrischilles Elizabeth A. , Cooper Cyrus , Lane Ann W. , Riggs B. Lawrence
Published: February 2010

Osteoporosis is widely viewed as a major public health concern, but the exact magnitude of the problem is uncertain and likely to depend on how the condition is defined. Noninvasive bone mineral measurements can be used to define a state of heightened fracture risk (osteopenia), or the ultimate clinical manifestation of fracture can be assessed (established osteoporosis). If bone mineral measurements more than 2 standard deviations below the mean of young normal women represent osteopenia, then 45% of white women aged 50 years and over have the condition at one or more sites in the hip, spine, or forearm on the basis of population-based data from Rochester, Minnesota. A smaller proportion is affected at each specific skeletal site: 32% have bone mineral values this low in the lumbar spine, 29% in either of two regions in the proximal femur, and 26% in the midradius. Although this overall estimate is substantial, some other serious chronic diseases are almost as common. More importantly, low bone mass is associated with adverse health outcomes, especially fractures. The lifetime risk of any fracture of the hip, spine, or distal forearm is almost 40% in white women and 13% in white men from age 50 years onward. If the enormous costs associated with these fractures are to be reduced, increased attention must be given to the design and implementation of control programs directed at this major health problem.
Is heritability a risk factor for postmenopausal osteoporosis?
Authors: Hansen Marc Allan , Hassager Christian , Jensen Signe Birk , Christiansen Claus
Published: February 2010

We investigated heritability as a risk factor for the development of osteoporosis in two randomly selected populations of postmenopausal women and their premenopausal daughters. We determined the familial resemblance in bone mass at three sites; the distal forearm, lumbar spine, and proximal femur, premenopausally and with increasing maternal postmenopausal age. We also examined the bone mass of daughters in relation to mothers with and without osteoporotic fractures. Peak bone mass among premenopausal siblings was significantly correlated at all sites (r = 0.30–0.42, p < 0.001). The same levels of resemblance were found between early postmenopausal mothers and premenopausal daughters. There was no significant difference in bone mass at any skeletal site between daughters of women with either peripheral or spinal fractures and daughters of women without fractures. We also examined familial resemblance with four biochemical markers of bone turnover (fasting urinary calcium and hydroxyproline, both corrected for creatinine, serum alkaline phosphatase, and plasma bone Gla protein). A generally significant resemblance were seen in premenopausal siblings (r = 0.25–0.39, hydroxyproline NS), but not between premenopausal daughters and postmenopausal mothers. We conclude that peak bone mass is hereditary in the distal forearm, lumbar spine, and proximal femur, but the mother-daughter resemblance explains only about 16% of the variability in daughters' bone mass. Furthermore, daughters of women with a moderate state of osteoporotic fractures are not substantially at an increased risk of having a low peak bone mass compared to the daughters of women without fractures.
Effects of deflazacort on aspects of bone formation in cultures of intact calvariae and osteoblast-enriched cells
Authors: Dr. Canalis Ernesto , Avioli Louis
Published: February 2010

Deflazacort, a synthetic glucocorticoid reported to have bone-sparing properties in vivo, and Cortisol were compared for their effects on bone formation in vitro. Deflazacort and Cortisol were studied for their effects on DNA and collagen synthesis in cultures of intact fetal rat calvariae and of osteoblast-enriched (Ob) cells from 21- to 22-day-old fetal rat parietal bone. Both steroids were also examined for their effects on skeletal insulin-like growth factor (IGF) I production, which is decreased by Cortisol and appears relevant to its mode of action. After 24 h of culture, deflazacort and Cortisol had limited effects on the parameters studied, although Cortisol at 100 nM decreased [3H]proline incorporation into collagen in intact calvariae. In contrast, after 72 h deflazacort and Cortisol at 1–100 nM inhibited the incorporation of [3H]thymidine into DNA and at 100 nM decreased the incorporation of [3H]proline into collagen and noncollagen protein in intact calvariae. Deflazacort and Cortisol at 10–1000 nM decreased calvarial collagen degradation to a similar extent. Both steroids had a similar activity, and at 100 nM for 72 h they decreased IGF-I production by calvariae; however, Cortisol at 10 nM was somewhat more effective than deflazacort in decreasing IGF-I levels. Deflazacort and Cortisol had analogous effects in Ob cell cultures. After 24 h of treatment, deflazacort at 100–1000 nM and Cortisol at 10–1000 nM decreased the labeling of DNA, and both steroids at 100–1000 nM caused a similar decrease in [3H]proline incorporation into collagen in Ob cells. In addition, deflazacort and Cortisol at 100 nM decreased IGF-I levels by about 60% in Ob cell cultures. In conclusion, deflazacort and Cortisol have similar inhibitory actions on bone DNA and collagen synthesis, collagen degradation, and IGF-I concentrations in vitro.
Expression of helix-loop-helix regulatory genes during differentiation of mouse osteoblastic cells
Authors: Murray Samuel S. , Glackin Carlotta A. , Winters Katherine A. , Gazit Dan , Kahn Arnold J. , Murray Elsa J.
Published: February 2010

Although much is known about the hormonal regulation of osteoblastic cell differentiation, much less is known about the nuclear regulatory molecules that affect this process. We analyzed the expression of several regulatory molecules of the helix-loop-helix (H-L-H) group in primary mouse calvarial cells and in MC3T3-E1 mouse osteoblastic cells in situations representing different degrees of cellular differentiation. H-L-H class regulators are known to participate directly in directing cell fate and differentiation decisions in other mesodermal lineages. Two of the molecules that we studied, Id and E12, have well-established roles in this process. The other, mTwi, the murine homolog of the Drosophila twist gene, is a newly cloned mammalian H-L-H gene. Levels of E12 RNA remained unchanged during differentiation. On the other hand, in both primary osteoblastic cells and MC3T3-E1 cells, the abundance of Id and mTwi declined with cell maturation; mTwi less dramatically than Id. That Id expression is causally related to differentiation is suggested by the finding that MC3T3-E1 cells transfected with an Id-expression plasmid fail to undergo differentiation. We conclude that helix-loop-helix regulatory genes are expressed in mouse osteoblastic cells, where they are likely to participate in differentiation. The E12 gene product is likely to function as a positive modulating factor. In contrast, Id inhibits differentiation, probably by sequestering other H-L-H gene regulators, including E12, in inactive complexes. The precise role of mTwi is more speculative at this time, but the observed pattern of expression is consistent with a role in early and midmesodermal specification that is terminated as cells differentiate.
Lateral spine densitometry is a more sensitive indicator of glucocorticoid-induced bone loss
Authors: Dr. Reid Ian R. , Evans Margaret C. , Stapleton Joanne
Published: February 2010

Osteoporosis is a common complication of glucocorticoid therapy. Bone density measurement is now commonly used in assessing which steroid-treated patients require specific interventions to reduce fracture risk. The recently developed techniques for the measurement of bone mineral density (BMD) of the vertebral body alone, by dual-energy x-ray absorptiometry (DXA) in the lateral projection, may be particularly useful in this context since steroid-induced bone loss is most marked in trabecular-rich regions like the vertebral body. This possibility has been assessed in the present study by the measurement of BMD in the lateral and anterioposterior (AP) projections in 28 women receiving chronic glucocorticoid treatment. The two BMD measurements were significantly related (r = 0.62, p < 0.001). When expressed in relation to age-appropriate normal values, lateral BMDs were lower than AP BMDs both in percentage terms (70.8 ± 4.4 versus 90.3 ± 2.6%, p < 0.001) and in terms of Z scores (-1.42 ± 0.22 versus −0.91 ± 0.24, p = 0.027). AP BMD Z scores classified 12 patients as osteopenic, whereas a further 7 were so categorized by lateral BMD Z score. It is concluded that lateral DXA scanning is a more sensitive indicator of glucocorticoid-induced osteopenia than conventional BMD measurement in the AP projection.
Protein kinase C modulates parathyroid hormone- but not prostaglandin E2-mediated stimulation of cyclic AMP production via the inhibitory guanine nucleotide binding protein in UMR-106 osteosarcoma cells
Authors: Koch Helena M. , Muir Helen , Gelderblom Dalene , Hough Stephen
Published: February 2010

In UMR-106 osteosarcoma cells we found that PTH activated both the cAMP/protein kinase A and the Ca2+-dependent phosphoinositide/protein kinase C (PKC) pathways, but prostaglandin E2 (PGE2) activated only the cAMP pathway. Activation of PKC by the phorbol ester PMA had no effect on cAMP production but enhanced PTH-stimulated cAMP production by 50% or more; the effect on PGE2-induced cAMP was negligible. Inhibition of the α-subunit of the inhibitory guanine nucleotide binding protein (Gi) by pertussis toxin pretreatment also enhanced PTH-mediated cAMP production but had no effect on PGE2-induced cAMP production. These results suggest that although PTH-mediated adenylate cyclase activity is regulated via both the stimulatory (Gs) and inhibitory (Gi) guanine nucleotide binding proteins, only Gs regulates PGE2-mediated adenylate cyclase activity in UMR-106 cells. Costimulation with pertussis toxin and PMA did not increase PTH-stimulated cAMP production above that obtained with PMA alone. This implies a similar target of action for pertussis toxin and PMA, that is, the α-subunit of Gi. The α-subunit of Gi was found to be a substrate for in vitro PKC phosphorylation of membrane fractions from UMR-106 cells, seen as a ±40 kD band on SDS-PAGE. Stimulation of in situ 32P-labeled cells with either PMA or PTH also enhanced incorporation of 32P into the 40 kD band. Using the peptide antisera AS/7 and EC/2, we showed that pertussis toxin-labeled subunits of both Gi1α/Gi2α and Gi3α could be immunoprecipitated, respectively, but immuinoprecipitation of membrane proteins after in situ phosphorylation and stimulation with PMA precipitated only Gi2α. We therefore conclude that modulation of adenylate cyclase activity by phorbol esters in UMR-106 osteosarcoma cells can be ascribed, at least in part, to PKC-mediated phosphorylation of the α-subunit of the Gi2 component of the adenylate cyclase regulatory complex.
Reduced vertebral bone density in hypercalciuric nephrolithiasis
Authors: Pietschmann Flavia , Breslau Neil A. , Pak Charles Y.C.
Published: February 2010

Dual-energy x-ray absorptiometry and single-photon absorptiometry were used to determine bone density at the lumbar spine and radial shaft in 62 patients with absorptive hypercalciuria, 27 patients with fasting hypercalciuria, and 31 nonhypercalciuric stone formers. Lumbar bone density was significantly lower in patients with absorptive (-10%) as well as in those with fasting hypercalciuria (-12%), with 74 and 92% of patients displaying values below the normal mean, whereas only 48% of the nonhypercalciuric stone formers had bone density values below the normal mean. In contrast, radial bone density was similar in all three groups of renal stone formers investigated. The comparison of urinary chemistry in patients with absorptive hypercalciuria and low normal bone density compared to those with high normal bone density showed a significantly increased 24 h urinary calcium excretion on random diet and a trend toward a higher 24 h urinary uric acid excretion and a higher body mass index in patients with low normal bone density. Moreover, among the patients with absorptive hypercalciuria we found a statistically significant correlation between the spinal bone density and the 24 h sodium and sulfate excretion and the urinary pH. These results gave evidence for an additional role of environmental factors (sodium and animal proteins) in the pathogenesis of bone loss in absorptive hypercalciuria. In conclusion, our data suggest an osteopenia of trabecular-rich bone tissues in patients with fasting and absorptive hypercalciurias.
Measurement of serum osteocalcin with a human-specific two-site immunoradiometric assay
Authors: Garnero Patrick , Grimaux Marc , Demiaux Brigitte , Preaudat Corinne , Seguin Patrick , Dr. Delmas Pierre D.
Published: February 2010

We developed a sensitive and specific two-site radioimmunoassay (IRMA) for human osteocalcin using human osteocalcin as a standard and two monoclonal antibodies raised against human osteocalcin purified from human cortical bone, a solid-phase anti-25–37 region and a tracer anti-5–13 sequence of the molecule. A wide range of osteocalcin levels (up to 300 ng/ml) can be measured with a sensitivity of 0.4 ng/ml. The intra- and interassay coefficients of variation are less than 4 and 6%, respectively. The recovery of human osteocalcin from serum samples ranges from 96 to 103%. IRMA was linear for serial sample dilutions in a wide range of serum osteocalcin levels, even in patients with chronic renal failure on hemodialysis. Depletion of serum in intact osteocalcin demonstrated that IRMA detects, in addition to the intact peptide, a large N-terminal midregion fragment that represents about 50% of total osteocalcin levels in normals and patients with Paget's disease and up to 75% in patients with chronic renal failure. This large fragment, previously unrecognized because it cannot be distinguished from intact osteocalcin with gel filtration chromatography, is not generated in vitro by incubation of the serum up to 26 h. We measured osteocalcin in the serum of 309 healthy adults (180 men and 129 women, age range 20–95 years), 36 patients with Paget's disease, 12 patients with primary hyperparathyroidism, 70 patients with chronic renal failure on hemodialysis, and 10 patients on corticosteroid therapy, simultaneously with human IRMA and with a conventional radioimmunoassay (RIA) based on bovine reagents. A tight correlation (r = 0.889) was observed between the two assays in the normal population, but the values obtained with IRMA were about threefold higher (mean 23.3 ± 10.5 versus 7.5 ± 3.4 ng/ml) than those obtained with RIA. Reported as Z scores, that is, number of standard deviations from the predicted normal mean adjusted for sex and age, these two assays (IRMA and RIA) gave concordant results in patients with Paget's disease (4.05 ± 6.21 versus 2.41 ± 2.53), primary hyperparathyroidism (4.14 ± 7.17 versus 2.13 ± 2.28), chronic renal failure (25.32 ± 24.49 versus 6.93 ± 5.48), and glucocorticoid treatment (-1.48 ± 0.78 versus −1.11 ± 0.57). However, IRMA was more discriminant from controls for all these metabolic bone diseases because the absolute values of mean Z scores with IRMA were significantly higher than those obtained with the RIA (p < 0.05–0.0001). We conclude that this new human-specific IRMA of osteocalcin may be more sensitive than bovine RIA for the clinical investigation of metabolic bone diseases.
Osteoporosis in anorexia nervosa: The influence of peak bone density, bone loss, oral contraceptive use, and exercise
Authors: Seeman Ego , Szmukler George I. , Formica Carmelo , Tsalamandris Con , Mestrovic Robert
Published: February 2010

Anorexia nervosa occurs early in life and predisposes to osteoporosis. Exercise may be protective. We asked: (1) Does failure to attain peak bone density contribute to the deficit in bone density? (2) Does oral contraceptive use protect against osteoporosis? (3) Is any protective effect of exercise confined to weight‐bearing sites? Areal bone density (g/cm2) and body composition were measured by dual x‐ray absorptiometry in 65 patients with anorexia nervosa and 52 controls. Comparing the 12 patients with primary amenorrhea and the 37 patients with secondary amenorrhea, bone density (mean ± SEM) at the lumbar spine was 0.88 ± 0.04 versus 1.06 ± 0.03 (P = 0.001), respectively. Bone density at the femoral neck was 0.80 ± 0.04 versus 0.92 ± 0.03 (P < 0.05), respectively. These values differed before, but not after, adjusting for the respective duration of illness (73.0 ± 10.3 versus 34.1 ± 4.8 months, P < 0.001) and fat‐free mass (31.6 ± 1.3 versus 35.4 ± 0.5 kg, P < 0.01). Bone density at the lumbar spine in the 16 patients with 31.8 ± 8.3 months of contraceptive exposure was higher than in the 49 patients with no contraceptive exposure (1.14 ± 0.05 versus 1.02 ± 0.02 P < 0.02) but was lower than in controls (1.14 ± 0.05 versus 1.27 ± 1.02, P < 0.01). No protective effect of contraceptive exposure was detectable at the femoral neck. Nevertheless, bone density at both sites correlated positively with the duration of contraceptive exposure (lumbar spine, R 0.63, P < 0.02; femoral neck, R = 0.66 P < 0.01) and negatively with duration of amenorrhea (lumbar spine, R = −0.53, P < 0.01; femoral neck, R = −0.49, P < 0.01, respectively). In the 19 patients who exercised vigorously, bone density was higher than in the 30 sedentary patients at the femoral neck (0.97 ± 0.04 versus 0.85 ± 0.03, P < 0.05), but not at the lumbar spine. Anorexia nervosa of earlier onset is likely to impose a greater fracture risk because of larger deficits in bone density. The deficits are more likely to be due to bone loss than low peak bone density, although a contribution to the deficit by low peak bone density cannot be excluded. Bone density of the lumbar spine may be more sensitive to estrogens than bone density of the proximal femur, and the proximal femur may be more sensitive to weight‐bearing exercise. Because partial protection against osteoporosis may be obtained at the spine using oral contraceptives and at the proximal femur by weight bearing, these modalities may warrant consideration as part of the treatment of this illness.
Journal of Bone and Mineral Research
Authors: Lanyon Lance E.
Published: February 2010

The continuing ability of the skeleton to withstand functional loads without damage requires that bone mass and architecture are adjusted according to the loads experienced. Load bearing is the only functional influence that requires a particular bone architecture, and functionally engendered strains within the bone tissue provide the only feedback containing the necessary information on the relationship between current architecture and prevailing load history. The specific strain-related objectives of the adaptive modeling and remodeling response to load bearing have not been adequately defined. They appear to be different for cortical and cancellous bone and vary according to cortical location. Experiments suggest that adaptive modeling and remodeling is sensitive to dynamic but not static strain change and that the osteogenic response to a period of dynamic strain is quickly saturated but is higher when the rate of change in strain is high and the distribution of strain unusual. Presumably it is the cumulative effect of this osteogenic response to load bearing that normally maintains bone mass above that seen in disuse situations. Through their independent effects on bone cell behavior, nutritional and hormonal factors can enable, enhance, limit, or frustrate full expression of the osteogenic response to strain change. However, such systemic factors do not appear to be able to engender or successfully imitate the sustained cumulative local response to load bearing that normally maintains functionally appropriate bone mass and architecture. Experiments in vivo and in vitro suggest that in osteocytes and surface osteoblasts the almost immediate response to strain change is increased production of prostacyclin. Surface osteoblasts also produce prostaglandin E. Only 5 minutes after loading, glucoses-phosphate dehydrogenase activity in osteocytes is increased in a local strain magnitude-related manner, and 24 h later there is an increase in osteocyte RNA. Exogenous PGE2 and PGI2 imitate the G6PD response in both osteocytes and osteoblasts, but only PGI2 imitates the loading-related increase in RNA in these two cell types. Indomethacin reduces the osteogenic response to loading in vivo and both the G6PD and RNA responses to loading in vitro. We hypothesize that the strain-related increase in PGE influences the synthetic activity of surface bone cells directly, whereas the strain-related increase in PGI2 additionally influences modeling and remodeling through the production of a cytokine or growth factor for which the loading-related RNA is coded. It may be at the stage of cytokine interaction that the potentially competing or complementary effects on modeling and remodeling of the loading and hormonal environments is resolved.
Journal of Bone and Mineral Research
Authors: Müller-Gerbl Magdalena , Putz Reinhard , Kenn Rolf
Published: February 2010

Since the work of Pauwels and his successors, it has been possible to use the distribution of subchondral bone density within a joint surface as a metric parameter that can reflect the principal long-term stress acting upon a joint. However, the x-ray densitometry method he employed cannot be applied to living people. A procedure was therefore developed whereby CT osteoabsorptiometry (CT OAM), based on the use of computed tomography, allows the distribution pattern of the density to be demonstrated in living subjects. This method has now been further developed, so that the form of the individual joint surfaces can be included by means of a three-dimensional reconstruction program. This method is presented here. In addition, selected representative examples of various joints from normal people, athletes, and patients are used to demonstrate the use of CT OAM. In these examples from living subjects, regularly occurring, reproducible distribution patterns of subchondral bone density can be recorded, reflecting changes in mechanical stresses on a joint (increased stress, reduced stress, and disorders of joint mechanics). CT osteoabsorptiometry is demonstrated as a suitable noninvasive technique for investigating the individual long-term stresses (loading history) acting on a living joint.
Physicochemical effects of acidosis on bone calcium flux and surface ion composition
Authors: Bushinsky David A. , Wolbach Wendy , Sessler Nelson E. , Mogilevsky Radion , Levi-Setti Riccardo
Published: February 2010

Net calcium flux (JCa) from bone in vitro is pH dependent. When pH falls below 7.40, through a reduction in [HCO3−], there is both physicochemical and cell-mediated JCa. To characterize the physicochemical effect of acidosis on bone we inhibited the bone-resorbing cells (osteoclasts) with the specific inhibitor calcitonin and studied the effect of acidosis on JCa and bone ion composition using an analytic high-resolution scanning ion microprobe. Neonatal mouse calvariae were cultured for 48 h in physiologically neutral pH medium (Ntl, pH = 7.41, [HCO3−] = 25 nM) or in medium that modeled metabolic acidosis (Met, pH = 7.10, [HCO3−] = 12), each with or without calcitonin (CT, 3 × 10−9 M). There was net calcium efflux in Ntl (JCa = 631 ± 36 nmol per bone per 48 h), which increased in Met (1019 ± 53, p < 0.01); CT inhibited JCa in Ntl (-54 ± 11, p < 0.01 versus Ntl), which increased in Met (197 ± 15, p < 0.01 versus Ntl + CT). In the presence of CT the increase in JCa in Met versus Ntl represents physicochemical bone dissolution. The Ntl bone surface (∼2 nm in depth) was rich in Na compared to Ca (Na/Ca = 11.9, counts/s of detected secondary ions), which fell in Met (Na/Ca = 6.0, p < 0.05); CT caused a further reduction of Na/Ca (3.1, p < 0.01 versus Ntl and versus Met), which was not altered in Met (2.6, p ≥ 0.05 versus Ntl + CT). The subsurface of the Ntl bone, eroded to a depth of = 100 nm with the microprobe, was also rich in Na/Ca (11.0), which fell in Met (4.1, p < 0.05); CT caused a further fall in Na/Ca (3.8, p < 0.01 versus Ntl), which again was not altered in Met (3.5, p ≥ 0.05 versus Ntl + CT). Acidosis causes a release of bone Ca and a fall in the ratio of bone Na/Ca, indicating a greater release of Na. Inhibition of osteoclastic function with calcitonin causes an influx of Ca to bone and a marked fall in bone Na/Ca, indicating little change in Na. Acidosis plus calcitonin causes Ca release with no change in Na/Ca, indicating that physicochemical bone mineral dissolution causes relatively equal Ca and Na release. Thus, the cell-mediated effect of acidosis-induced bone resorption appears responsible for the excess Na efflux.
Measurement of osteoclasts and bone resorption by automated image analysis
Authors: Dunstan Colin R. , Lauren Peter D. , Somers Nicole M. , Evans Richard A.
Published: February 2010

An automated image analysis method is described for measurement of osteoclasts and resorbing surface in calcified bone. Osteoclasts, osteoid, and mineralized bone were measured in a single section, reacted for acid phosphatase activity, and then stained with orange G and light green stain. Three images were acquired of each field with a monochromatic camera at illuminating wavelengths of 635, 540, and 480 nm (selected using a stage monochromator). These wavelengths were chosen according to the absorption spectra of the different image components to maximize absorption differences between osteoclasts (red), mineralized bone (blue/green), and osteoid (orange). These components were then discriminated according to operator-defined ranges of color density (mineralized bone) or color fraction (osteoid and osteoclasts). A gray level coded segmented image was produced, from which was determined the area, perimeter, and number of each component and the length of contact zones with the marrow and between these components. The method was evaluated by twice measuring 10 bone sections from patients with end-stage liver failure awaiting liver transplantation. The method was quite reproducible, with coefficients of variation varying between 4% for bone volume (% tissue volume) and 22% for osteoid surface (% bone surface). The sections were also measured using a previously established semiautomated method. Coefficients of variation between methods were higher varying between 4% for bone volume (% tissue volume) and 56% for osteoid volume (% bone volume). The automated method gave a substantial time saving compared to the semiautomated method. An interactive technique was used in adjacent sections to evaluate tetracycline labeling and osteoblast surfaces.
Tumor necrosis factors α and β can stimulate bone resorption in cultured mouse calvariae by a Prostaglandin-independent mechanism
Authors: Lerner Ulf H. , Ohlin Acke
Published: February 2010

Human recombinant tumor necrosis factors α and β (TNF-α and TNF-β), at and above 1 ng/ml (≅ 70 pM), caused a dose- and time-dependent enhancement of 45Ca release from neonatal mouse calvarial bones in vitro. In addition, TNF-α and TNF-β (3–100 ng/ml) caused a dose-dependent stimulation of prostaglandin E2 (PGE2) formation in the calvarial bones. TNF-α also enhanced the biosynthesis of PGI2, as assessed by analysis of the stable breakdown product 6-keto-PGF1α. The stimulatory actions of TNF-α and TNF-β on PGE2 formation was maximal at 12 h. Indomethacin, flurbiprofen, and meclofenamic acid, three structurally unrelated nonsteroidal antiinflammatory drugs, abolished PGE2 biosynthesis induced by TNF-α and TNF-β (100 ng/ml). The 45Ca release stimulated by TNF-α and TNF-β (100 ng/ml), however, was only slightly reduced by indomethacin, flurbiprofen, and meclofenamic acid. The partial inhibitory effect of indomethacin on 45Ca release was seen over a wide range of TNF-α concentrations, without affecting the concentration producing half-maximal stimulatory response. TNF-α and TNF-β (100 ng/ml) stimulated bone matrix breakdown, as assessed by analysis of the release of 3H from bone prelabeled with [3H]proline. Also, the stimulatory effect of TNF-α and TNF-β on bone matrix degradation was partially reduced by indomethacin. Hydrocortisone (1 μM) and dexamethasone (0.1 μM) abolished TNF-α- and TNF-β-induced production of PGE2. In contrast to the cyclooxygenase inhibitors, the corticosteroids did not affect the stimulatory action by the cytokines on 45Ca release. These observations suggest that TNF-α and TNF-β can stimulate bone resorption in vitro by prostaglandin-independent mechanisms.
Modulation of vitronectin receptor-mediated osteoclast adhesion by Arg-Gly-Asp peptide analogs: A structure-function analysis
Authors: Horton Michael A. , Dorey Elaine L. , Nesbitt Stephen A. , Samanen James , Ali Fadia E. , Stadel Jeffrey M. , Nichols Andrew , Greig Russel , Helfrich Miep H.
Published: February 2010

This study details the investigation of induction of retractile shape change in the osteoclast through inhibition of adhesion between osteoclasts and matrix with (1) peptide analogs bearing an Arg-Gly-Asp (RGD) sequence, (2) antibodies to the integrin αvβ3 vitronectin receptor, and (3) the RGD-containing snake venom peptide echistatin. Osteoclast retraction on dentin has been demonstrated for GRGDSP peptide, in contrast to the inactivity of the analog containing the conservative RGE sequence modification. An osteoclast adhesion assay employing rat or chick bone cells and serum-coated glass coverslips as substrate was developed for routine evaluation of inhibition of adhesion. Antibodies F4 and F11 to the β3 chain of rat vitronectin receptor were effective at submicromolar concentrations in rat osteoclasts (IC50 0.29 and 0.05 μM, respectively), whereas MAb 23C6 to human/chick vitronectin receptor was somewhat less effective against chick osteoclasts (ICso 1.6 μM). A rank order of RGD analog activity (mean IC50, μM) in the serum-coated glass adhesion assay was derived for the linear peptides GRGDSP (201 μM), GRGDTP (180 μM), Ac-RGDS-NH2 (84 μM), Ac-RGDV-NH2 (68 μM), RGDV (43 μM), GRGDS (38 μM), and RGDS (26 μM). The two most potent short peptides were the cyclic analog SK&F 106760 Ac-S,S-cyclo-(Cys-(NαMe)Arg-Gly-Asp-Pen)-NH2 (IC50 7.0 μM), and the Telios peptide H-Gly-S,S-cyclo-(Pen-Gly-Arg-Gly-Asp-Ser-Pro-Cys)-Ala-OH (IC50 6.6 μM). The snake venom peptide echistatin was the most potent substance evaluated in the serum-coated glass assay (IC50 0.78 nM) employing either rat or chick osteoclasts. Specificity control peptides fibronectin CS1 (ligand for VLA-4), fibrinogen H12 (alternate ligand for gpIIb/IIIa), and Iaminin cell binding fragment YIGSR were inactive up to 800 μM. Comparison of SK&F 106760 and the Telios peptide as inhibitors of platelet aggregation (IC50 0.36 and 10.1 μM, respectively) and inhibitors of L8 skeletal muscle cell adhesion to vitronectin (IC50 67.2 and 12.3 μM, respectively) suggests that the Telios peptide is nonselective whereas SK&F 106760 may be selective with regard to β3 integrins. This study demonstrates that structural modification in RGD peptides and the use of antireceptor antibodies or the venom peptide echistatin yields potent inhibitors of vitronectin receptor-mediated adhesion in isolated rat and chick osteoclasts. It is hoped that further peptide modification will yield improved specificity and thus selective inhibitory effects upon bone resorption.
Subcellular localization and partial purification of the 25-hydroxyvitamin D3 1-hydroxylation reaction in the chick myelomonocytic cell line HD-11
Authors: Shany Shraga , Ren Song-Yang , Arbelle Jonathan E. , Clemens Thomas L. , Adams John S.
Published: February 2010

Hypercalcemia in human granuloma-forming diseases like sarcoidosis results from the endogenous overproduction of 1,25-dihydroxyvitamin D [1,25-(OH)2D] by disease-activated tissue macrophages. The recent identification of an immortalized chick myelomonocytic cell line, HD-11, that constitutively expresses a 25-hydroxyvitamin D (25-OHD) 1-hydroxylation reaction has alleviated dependence on studying primary macrophage cultures with no replicative potential in vitro. In these experiments we established conditions for the maximal expression of the HD-11 cell 25-OHD3-1-hydroxylation reaction and localized this activity to the mitochondrial fraction. On a per cell basis, the activity of HD-11 cell 25-OHD3 1-hydroxylation reaction was comparable to that in primary cultures of chick renal tubular epithelial cells, which express the authentic renal 25-OHD3 1-hydroxylase. Maximal product yield was achieved after incubation of HD-11 cells with 200 nM 25-OHD3 for 3 h. Although adherent monolayers possessed 3- to 4-fold more capacity for hormone production than cells in suspension, suspended cells exhibited easily detectable 25-OHD3 catalytic activity (0.58 ± 0.08 pmol per 106 cells per h; ± SEM), 50% of which remained solubilized in a sonicate of suspended cells cleared of nuclei and plasma membrane. Subcellular localization disclosed 91% of the residual activity to be concentrated in the mitochondrial subfraction. A detergent-solubilized extract of this mitochondrial subfraction contained 1.9 ± 0.3 pmol 1,25-(OH)2D3 synthetic capacity per mg protein. The catalytic activity (1-hydroxylase activity) was concentrated 20.2-fold after chromatography on octyl-amino agarose and was associated with 0.054 nmol cytochrome P450 per mg protein. These data suggest that HD-11 cell 25-OHD3 1-hydroxylation reaction may be similar to the well-characterized 25-OHD3 1-hydroxylase expressed in the kidney.
Rapid and sensitive method for quantitation of bone Gla protein mRNA using competitive polymerase chain reaction
Authors: Araki Nobuhito , Robinson Darlene F. , Nishimoto Satoru K.
Published: February 2010

A method for sensitive quantitation of bone gla protein (BGP, osteocalcin) mRNA has been developed using competitive polymerase chain reaction after reverse transcription (competitive RT-PCR). The complementary DNA (cDNA) reverse transcribed from sample RNA was coamplified in a PCR with a known amount of mutant BGP cDNA (competitor) using the identical oligonucleotide primers. The mutant cDNA with its unique restriction site allowed quantitation of sample and mutant PCR products after densitometric analysis of ethidium bromide-stained agarose gels. A linear relationship between initial sample BGP amount and the ratio of BGP to mutant BGP band intensity was obtained and used to make a standard curve to determine the initial BGP mRNA of unknown samples. These standard curves were made with known amounts of recombinant BGP cDNA. The competitive RT-PCR for BGP allows measurement of twofold differences in 1 and 2 μg total RNA and requires at least 10 times less sample RNA than usual Northern blotting. Moreover, heteroduplexes with one BGP strand and one mutant BGP strand formed as a result of high PCR cycles were quantifiable. This provided the advantages of rapid quantitation from ethidium bromide-stained gels without blotting, hybridization, or autoradiography. Multiple samples could be assayed for greater confidence in the results. The sensitivity, accuracy, and ease of the assay will facilitate analysis of BGP mRNA from a small amount of sample. The assay has been used to confirm the BGP mRNA changes with hormonal treatment in cultured cells and the age-related changes in whole tibia in vivo.
Ultrastructural immunolocalization of noncollagenous (osteopontin and osteocalcin) and plasma (albumin and α2HS-glycoprotein) proteins in rat bone
Authors: Dr. McKee Marc D. , Farach-Carson M.C. , Butler W.T. , Hauschka P.V. , Nanci A.
Published: February 2010

The high-resolution, postembedding protein A-gold immunocytochemical technique was used to visualize the distribution of two noncollagenous bone proteins, osteopontin (OPN) and osteocalcin (OC), and two plasma proteins, α2HS-glycoprotein (α2HS-GP) and albumin (ALB), in sections of Lowicryl K4M-embedded rat tibial and alveolar bone. In the primary spongiosa of the metaphysis, a seam of organic material (lamina limitans) that labeled intensely with OPN and OC antibodies was observed at the bone/calcified cartilage interface just below the zone of vascular invasion of the growth plate. With deposition of bone matrix proper by osteoblasts in this region and its subsequent mineralization, extensive areas of bone were heavily labeled with anti-OPN, anti-OC, and anti-α2HS-GP antibodies, where the majority of gold particles were associated with amorphous, electron-dense patches of organic material throughout the mineralized bone. In the unmineralized osteoid, substantially less labeling was observed, and where occasional mineralization loci were dispersed throughout the osteoid layer, these sometimes showed a concentration of gold particles. ALB labeling, on the other hand, was moderate and generally diffuse throughout the mineralized bone matrix and the osteoid. In alveolar bone, labeling patterns were generally similar to those found in tibial bone. Particularly striking in alveolar bone, however, was an intense anti-OPN labeling of (1) the lamina limitans at cell-lined bone surfaces, including that surrounding cell processes and osteocytes, (2) cement (reversal, resting) lines, and (3) the perilacumar matrix of some osteocytes. In summary, these data suggest that certain plasma proteins, such as α2HS-GP, interact with bone matrix proteins, such as OPN and OC, at sites of tissue mineralization and that the presence of OPN in mineralized bone and at bone surfaces (lamina limitans) and cement lines has a multifunctional role, including regulation of mineralization and mediation of cell dynamics during endochondral and intramembranous bone modeling and remodeling.
Ontogenesis of ultrastructural features during osteogenic differentiation in diffusion chamber cultures of marrow cells
Authors: Passi-Even L. , Gazit D. , Bab I.
Published: February 2010

Three stages of osteogenic differentiation can be identified in in vivo diffusion chamber cultures (DCC) of unselected marrow cells, namely, proliferation, differentiation, and maturation (mineralization). These stages were characterized correlatively by in situ differential cell counts, alkaline phosphatase activity, and mineral accumulation. In the present study, the ultrastructure of marrow cell DCC was examined after incubation for 3–21 days. Features characteristic of osteoblastic and chondroblastic differentiation were first noted in 12 day DCC. Sites of osteoblastic differentiation showed cell-cell contacts associated with an increased cell density. The osteoblastic cells had long processes and were embedded in matrix with prominent fiber bundles reminiscent of collagen type I. The chondroblastic cells appeared solitary in areas of lesser cell density. By contrast to the long osteoblastic cell processes, they had short plasmalemmal projections and the matrix surrounding them contained single, thin, short fibers reminiscent of collagen type II, as well as proteoglycan granules. Both cell types showed prominent cytoskeletal elements, rough endoplasmic reticulum, and Golgi. One finding, previously unnoted in differentiating osteogenic cells, was mitochondria with condensed cristae that represent an increased rate of energy metabolism. These mitochondria were particularly abundant in the differentiation stage and declined as the cultures matured. These findings, together with previous reports in the epiphyseal growth plate, suggest that mineralization is associated with an optimal level of energy metabolism rather than extreme hypo- or hyperoxia. The set of ultrastructural parameters defined here in the marrow cell DCC may serve as useful markers for cells undergoing osteogenic differentiation.
Immunoassay of pyridinoline crosslink excretion in normal adults and in paget's disease
Authors: Delmas Pierre D. , Gineyts Evelyne , Bertholin Annick , Garnero Patrick , Marchand François
Published: February 2010

A new immunoassay (ELISA) based on an antiserum that preferentially recognizes the free form of the pyridinoline (Pyr) crosslink was used to assess the urinary excretion of Pyr in a large normal sample of the population comprising 236 men and 193 women 30–90 years of age. Urinary Pyr increased significantly with age in both sexes. In women Pyr was higher than in men (57 ± 21 versus 46 ± 17 nmol/mmol creatinine, p < 0.001), and the menopause was reflected by a mean 37% increase, from an average 43 to 59 nmol/mm creatinine (p < 0.001). In 52 patients with active Paget's disease of bone, Pyr excretion was markedly increased (206 ± 160 nmol/mmol creatinine, p < 0.001 versus controls) and decreased by 71% after 3 days of IV treatment with the bisphosphonate pamidronate. Free Pyr measured by the ELISA and the total urinary excretion measured by HPLC were highly correlated both in normals (n = 74, r = 0.83, p < 0.001) and in Pagetic patients (n = 20, r = 0.93, p < 0.001). The mean intra- and interassay coefficients of variation of the ELISA were below 9 and 15%, respectively. It is concluded that this new convenient immunoassay of Pyr should be valuable for the clinical investigation of patients with osteoporosis and other metabolic bone diseases.
Determinants of bone mineral density and risk factors for osteoporosis in healthy elderly women
Authors: Ooms Marcel E. , Lips Paul , van Lingen Arthur , Valkenburg Hans A.
Published: February 2010

Bone mineral density (BMD) and risk factors for osteoporosis were assessed in 348 apparently healthy women over 70 years of age (mean 82.3 years). BMD was measured at both hips and the dominant distal radius. With stepwise multiple regression the best determinants of BMD, selected from anthropometric measurements, age, and years since menopause, were body weight and years since menopause (R2 between 0.07 and 0.20, p < 0.001). Risk and protective factors for osteoporosis were analyzed as indicator variables by multiple regression and corrected for confounding by age, years since menopause, and body weight. Significantly lower BMD at the hip was found in participants with impaired mobility (-5%) and users of loop diuretics (-5%). Use of thiazide(like) diuretics did not influence BMD significantly at any site. Users of oral corticosteroids had a significantly lower BMD at the hip and the distal radius (range -9.1 to -24.3%). Participants with a history of Colles' fracture (n = 56) had a significantly lower BMD at the other radius (-12.9%). The mean calcium intake from dairy products was high (mean 921 mg/day), only 11% having an intake below 500 mg. A relation of calcium intake with BMD could not be detected at any measurement site. We conclude that BMD cannot be adequately predicted in elderly women. Risk factors for low BMD in the elderly are low body weight, high number of years since menopause, impaired mobility, and use of loop diuretics and oral corticosteroids. Calcium intake was not a risk factor in this study, but the number of individuals on a low calcium intake was small.
Flutamide-mediated androgen blockade evokes osteopenia in the female rat
Authors: Dr. Goulding Ailsa , Gold E.
Published: February 2010

Androgens are believed to play a role in building and maintaining bone in the female, as well as in the male. The antiandrogen drug flutamide inhibits responses to androgens from both the gonads and the adrenals. Antiandrogens prevent androgens stimulating bone cell proliferation and differentiation in vitro, but effects of androgen blockade on bone metabolism in vivo have not been tested. The present study was undertaken to determine whether androgen blockade with flutamide (15 mg/kg body weight orally daily) would influence bone turnover or bone composition (1) in female rats with intact ovaries and (2) in rats made estrogen-deficient with the luteinizing hormone releasing hormone (LHRH) agonist, buserelin (25 μg/kg body weight per day SC). Four groups of rats with 45Ca-labeled skeletons were studied for 4 weeks: group A, placebo; group B, buserelin; group C, flutamide; group D, flutamide + buserelin. Total-body calcium values (mean ± SD) were (mg) 2007 ± 109, 1779 ± 138 (P < 0.01 versus group A), 1818 ± 140 (P < 0.01 versus group A), and 1690 ± 75 (P < 0.01 versus group A) in groups A-D, respectively. Thus both buserelin and flutamide induced osteopenia. Skeletal 45Ca changes suggested buserelin-mediated estrogen deficiency bone loss was due to increased bone resorption, but flutamide-mediated androgen deficiency bone thinning was caused principally by reduced bone formation. These findings support the view that androgens play an important role in preserving bone mass in the female rat. Importantly, adequate estrogen status did not compensate for flutamide-mediated osteopenia. Although this work was done in an animal model, it seems possible that antiandrogen drugs cause osteopenia in people as well as in the rat. Further study of the likely bone-protective effects of androgen are also warranted in women.