bluuebunny/crossref_metadata_2025 huggingface dataset:
Pattern of gene expression following rat tibial marrow ablation
Authors: Suva Larry J. , Seedor Gregory J. , Endo Naoto , Quartuccio Helen A. , Thompson David D. , Bab Itai , Rodan Gideon A.
Published: February 2010

Following injury to bone marrow there is a phase of osteogenesis in which bone trabeculae replace the initial blood clot and fill the marrow cavity. The newly formed bone is subsequently fully resorbed by osteoclasts and normal bone marrow is restored. In this study we correlated the morphologic events with the pattern of gene expression that defines this sequence. Following marrow ablation, the trabecular bone volume in the affected section of the marrow cavity increased from control to 27% at day 6, declined to 18% at day 8, and eventually returned to control levels at day 14. Osteoblast number increased up to day 6 and declined substantially by day 8, but the number of osteoclasts peaked between days 8 and 10. Histologic analysis of alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) activity correlated with the observed cellular changes. Northern blot analysis of the levels of AP, osteocalcin (OC), and osteopontin (OP) mRNA shows a specific pattern of regulated gene expression, with AP mRNA maximal at day 6, OC mRNA very low until days 6–8, and OP mRNA expressed at very high levels throughout. In addition, procollagen α1(I) and α1(III) mRNAs show a regulated pattern of expression, with procollagen α1(I) maximally expressed between days 4 and 10 and procollagen α1(III) expressed at lower levels between days 4 and 6. The mRNA encoding insulin-like growth factor I (IGF-I) was found to be highly expressed between days 5 and 12; however, transforming growth factor β1, (TGF-β1) and TGF-β3 mRNA were only weakly expressed between days 4 and 10. These data demonstrate a temporal pattern of gene expression consistent with the observed morphologic profile, identify changes in growth factor mRNA that may be related to this repair process, and suggest that this is a suitable model for studying in vivo a synchronized sequence of bone formation and resorption at a well-defined anatomic site.
Protein kinase C-activating domains of parathyroid hormone-related protein
Authors: Dr. Gagnon Lyne , Jouishomme Hervé , Whitfield James F. , Durkin Jon P. , MacLean Susanne , Neugebauer Witold , Willick Gordon , Rixon Ray H. , Chakravarthy Balu
Published: February 2010

N-terminal fragments of PTH-related protein (PTHrP), PTHrP-(1–34), and PTHrP-(1–40) stimulated both adenylyl cyclase and a mechanism that increases membrane-associated protein kinase C (PKC) activity in ROS 17/2 rat osteosarcoma cells. There were two peaks in the PKC response to the N-terminal PTHrP fragments: one peak was obtained with picomolar and the other with nanomolar PTHrP concentrations. The PKC-stimulating picomolar concentrations of the PTHrP fragments did not detectably stimulate adenylyl cyclase, but the nanomolar concentrations did. Since a similar two-peak response of PKC activity was obtained with PTHrP-(28–34), the single, N-terminal PKC activation domain of the PTHrP is in the same 28–34 region of the molecule as that of PTH despite this region having different primary amino acid sequences in the two hormones. Unlike PTH, PTHrP has a second PKC activation domain, as indicated by the ability of picomolar concentrations of the PTHrP-(107–111) fragment to stimulate maximally membrane-associated PKC activity in the osteosarcoma cells.
Outcome of bone mineral density in anorexia nervosa patients 11.7 years after first admission
Authors: Herzog Wolfgang , Minne Helmut , Deter Christian , Leidig Gudrun , Schellberg Dieter , Wüster Christian , Gronwald Rainer , Sarembe Egbert , Kröger Friedebert , Bergmann Günther , Petzold Ernst , Hahn Peter , Schepank Heinz , Ziegler Reinhard
Published: February 2010

Osteopenia is a typical finding in patients suffering from anorexia nervosa. Unfortunately, available longitudinal studies are limited by a relatively short follow-up period. Therefore cross-sectional long-term follow-up studies may help to determine both the outcome of this bone lesion and variables that influence its subsequent development. Of an initial 66 consecutive patients with anorexia nervosa, 51 (77.3%) could be further evaluated. After an average of 11.7 years following first admission, cross-sectional measurements of lumbar and proximal radial bone mineral density (BMD) were performed. The ability to predict BMD using variables obtained from anamnestic and clinical data was then determined by multiple-regression analysis. The BMD of both radial and lumbar bone in anorexic patients with poor disease outcome (as defined by the Morgan-Russell general outcome categories) deviated by −2.18 and −1.73 SD (Z score), respectively. In patients with a good disease outcome lumbar BMD was significantly less reduced compared with radial BMD (–0.26 versus −0.68 SD). Variables reflecting estrogen deficiency and nutritional status in the course of the disease, that is, relative estrogen exposure (for lumbar BMD) and years of anorexia nervosa (for radial BMD), allowed the best prediction of BMD. A marked reduction in cortical and trabecular BMD in anorexic patients with poor disease outcome suggests a higher risk of fractures in these patients. Furthermore, the finding of a persistently reduced cortical and a slightly reduced trabecular BMD, even in patients with good disease outcome, suggests that a recovery of trabecular BMD might be possible, at least in part. Recovery of cortical bone, if possible at all, seems to proceed more slowly.
Procollagen synthesis and extracellular matrix deposition in MG-63 osteosarcoma cells
Authors: Jukkola Arja , Risteli Leila , Melkko Jukka , Dr. Risteli Juha
Published: February 2010

We compared the procollagen synthetic properties of MG-63 osteosarcoma cells with those of cultured human skin fibroblasts. In both cells, the expressions of type I and III procollagens are largely dependent on the constant presence of ascorbate and coordinately decreased by the neutral polymer dextran T-40. The amino-terminal propeptides of pro-α1 and pro-α2 chains of type I procollagen are phosphorylated and those of the pro-α1 and pN-α1 chains of type III procollagen both phosphorylated and sulfated, there being no difference in net charge in the propeptides between these cell types. The major differences between MG-63 and normal fibroblasts are the exceptionally high relative synthesis of type III procollagen by MG-63 cells, up to about 40% of the total of types I and III (6% in cultured skin fibroblasts), and the inability of ascorbate-supplemented MG-63 cells to deposit collagens into an insoluble pericellular matrix. A longer dextran treatment shifts up to one-fourth of the proline-labeled extracellular macromolecules into the matrix fraction within 4 days (in control 4%). Despite processing of the procollagens to the respective collagens in the matrix, neither control matrices nor those induced by dextran induced increased production of alkaline phosphatase. In cultures up to 4 days postconfluence the proportion of type III collagen produced tended to increase over that in early confluent cultures. With respect to collagen production, the MG-63 cell line is not a representative of the osteoblast lineage but rather resembles a proliferative wound fibroblast.
Additive effect of treadmill exercise and 17β-estradiol replacement on prevention of tibial bone loss in adult ovariectomized rat
Authors: Yeh James K. , Liu Chung C. , Aloia John F.
Published: February 2010

The effects of 17β-estradiol (E2) and treadmill exercise on tibial bone mass, tibial uptake of 45Ca, and proximal tibia osteoblast and osteoclast cell number were determined in adult ovariectomized rats. Female rats aged 10 months were ovariectomized and divided into five groups: (1) sham-operated; (2) ovariectomized; (3) ovariectomized, given 10 μg E2 biweekly; (4) ovariectomized, trained to exercise on a treadmill daily; and (5) ovariectomized, given E2 and exercised. E2 and/or exercise interventions were started 2 months following surgery and continued for 4 months. The calcium content of the tibial metaphysis and diaphysis and the proximal cancellous bone (BV/TV) were lower in the ovariectomized than in the sham-operated controls 6 months after ovariectomy. This lower bone content was associated with a greater bone uptake of 45Ca and a greater number of osteoblasts and osteoclasts in the proximal tibia compared to the control rats. The meta-physeal calcium content was higher and the 45Ca uptake and osteoblast and osteoclast number were lower in the E2-treated rats than in the nontreated rats. In the exercised group, higher diaphyseal calcium content and proximal cancellous bone were associated with lower bone resorption parameters without a significant effect on bone formation. This study demonstrates that E2 primarily influences tibial cancellous bone of the ovariectomized rat and a positive adaptation to exercise occurs in both cancellous and cortical bone. Under estrogen deficiency, E2 replacement suppresses increased bone formation and resorption; exercise suppresses mainly bone resorption. The effects of E2 replacement and exercise training are independent and additive.
Precision of dual-energy x-ray absorptiometry: Development of quality control rules and their application in longitudinal studies
Authors: Orwoll Eric S. , Oviatt Shelia K. , Biddle Julie A.
Published: February 2010

In research settings, longitudinal measurements of bone mineral density have become an integral part of the assessment of patients with metabolic skeletal disorders. To adequately utilize longitudinal measures, confidence in the long-term precision of the measurement technique must be very high. Dual-energy x-ray absorptiometry (DXA) has become commonly utilized in this context, and to better understand its long-term precision and to develop quality assurance protocols for its use, we examined the performance of eight DXA machines over a 3 year period. Anthropomorphic spine phantoms were measured frequently on each machine during the period of observation, and precision was estimated from the consistency of these determinations. Overall precision was excellent (mean longitudinal coefficient of variation, 0.4%). Nevertheless, by using a series of objective quality control criteria, small alterations in the performance of each machine were identified (mean number of changes, 4.6 in 3 years; mean magnitude, 0.0039 g/cm2, or 0.4%). The cumulative effects of those changes were sufficient to cause a significant (albeit minor) change in the regression slopes (phantom mineral density versus time) of most machines. The same quality control rules were also used to quantitate the magnitude of change and to adjust retrospectively machine performance during the period of observation, such that alterations were minimal and regression slopes were not significantly different from zero. Although the precision of DXA is excellent, alterations in machine function must be anticipated during longitudinal use. The development of quality control protocols provides the means to detect change objectively and to adjust for alterations in performance during the course of longitudinal evaluations.
Cytoplasmic pH influences cytoplasmic calcium in MC3T3-E1 osteoblast cells
Authors: Lin H.-S. , Hughes-Fulford M. , Kumegawa M. , Pitts A.C. , Snowdowne Kenneth W.
Published: February 2010

We found that the cytoplasmic concentration of calcium (Cai) of MC3T3-E1 osteoblasts was influenced by the type of pH buffer we used in the perfusing medium, suggesting that intracellular pH (pHi) might influence Cai. To study this effect, the Cai and pHi were monitored as we applied various experimental conditions known to change pHi. Exposure to NH4Cl caused a transient increase in both pHi and Cai without a change in extracellular pH (pHo). Decreasing pHo and pHi by lowering the bicarbonate concentration of the medium decreased Cai, and increasing pHi by the removal of 5% CO2 increased Cai. Clamping pHi to known values with 10 μM nigericin, a potassium proton ionophore, also influenced Cai: acid pHi lowered Cai, whereas alkaline pHi increased it. The rise in Cai appears to be very sensitive to the extracellular concentration of calcium, suggesting the existence of a pH-sensitive calcium influx mechanism. We conclude that physiologic changes in pH could modulate Cai by controlling the influx of calcium ions and could change the time course of the Cai transient associated with hormonal activation.
TGF-β1 induces bone closure of skull defects: Temporal dynamics of bone formation in defects exposed to rhTGF-β1
Authors: Beck L. Steven , Amento Edward P. , Xu Yvette , Deguzman Leo , Lee Wyne P. , Nguyen Tue , Gillett Nancy A.
Published: February 2010

The temporal dynamics of bone repair in a skull defect in rabbits was examined to characterize the in vivo cellular events occurring following a single local application of recombinant human TGF-β1 (rhTGF-β1). Rabbits received vehicle or 0.4, 1, 2, or 5 μg rhTGF-β1 applied to 12 mm defects at the time of surgery. The defect sites were subsequently evaluated by radiography and qualitative and quantitative histology at time points ranging from 1 to 180 days. Based on radiographic assessment, the defect area decreased rapidly in a dose-dependent manner through 35 days after surgery in the rhTGF-β1-treated groups. Minimal closure occurred in sites administered vehicle control at all time points examined. Sites treated with rhTGF-β1 were characterized histologically by an increase in parameters of active bone formation through 49 days, including percentage osteoid surface, percentage osteoblast/total surface, and an increase in the trabecular bone volume. Bone resorption parameters were increased at 16 and 49 days with histologic evidence of remodeling from woven to lamellar bone. By 70 days, no differences were observed among the groups for parameters of either bone formation or resorption. Bone formation rate was not altered with rhTGF-β1 treatment at any time point. These results indicate that exogenously applied rhTGF-β1 stimulated the recruitment and proliferation of osteoblasts at the defect site, resulting in a rapid deposition of bony matrix, with normal remodeling processes occurring thereafter. This study supports the hypothesis that TGF-β1 is a potent osteoinductive growth factor in vivo and may have potential application as a therapeutic aid to nonhealing bony defects.
Bone and mineral metabolism in the androgen-resistant (testicular feminized) male rat
Authors: Dr. Vanderschueren D. , van Herck E. , Suiker A.M.H. , Visser W.J. , Schot L.P.C. , Chung K. , Lucas R.S. , Einhorn T.A. , Bouillon R.
Published: February 2010

Androgens have important effects on bone in vivo, possibly by direct activation of the androgen receptors in osteoblasts. To test this hypothesis, calcium homeostasis, bone mass, and bone turnover were evaluated in mature (4-month-old) androgen-resistant (testicular feminized, TFM) male rats. Data were compared with data from both female and male littermates of the same age and strain. Compared to normal males, TFM had similar serum testosterone, twofold higher estradiol and estrone, and sixfold higher androstenedione concentrations. Compared to normal females, TFM rats showed lower estradiol but also elevated concentrations of androstenedione and estrone. Despite similar free 1,25-(OH)2D3 concentrations, both TFM and male rats maintained higher serum calcium and phosphate concentrations than their female littermates. Serum IGF-I concentrations in TFM rats were decreased compared to male rats (-12%) or female rats (-27%). Serum osteocalcin concentrations, however, were twofold higher in TFM rats than in females but not significantly different from males. Femoral length, diameter, and cortical thickness were intermediate between those of males and females. The cancellous bone density of the femur and cancellous bone volume of the proximal metaphysis of the tibia, however, were not significantly different between groups. The ash weight of the tibia was also not significantly different, and the ash weight of the four distal lumbar vertebrae ranged between male and female values. Bone mechanical properties as measured by torsional strength and energy absorption of the femur were lower in TFM than in females but not different from males. Osteoblast surfaces, osteoid, and osteoclast surfaces in the proximal tibial metaphysis of TFM rats were in the female range and lower than in males. Bone formation and mineral apposition rates measured at the same site were intermediate between male and female rates. Bone formation rates were significantly higher in male than in female rats. We conclude that the absence of functional androgen receptors results in a decrease in radial and longitudinal bone growth and in a decrease in serum IGF-I concentrations. TFM rats, however, have a cancellous bone volume and density similar to those of their normal male and female littermates. Bone turnover at the cancellous level is not increased compared to normal males. Cancellous bone volume in androgen resistance, in contrast to androgen deficiency, is probably maintained by a modest increase in serum estrogen concentrations.
Stimulation of interleukin-6 production by either calcitonin gene-related peptide or parathyroid hormone in two phenotypically distinct bone marrow-derived murine stromal cell lines
Authors: Sakagami Y. , Girasole G. , Yu X.-P. , Boswell H.S. , Manolagas Stavros C.
Published: February 2010

It was recently shown that interleukin-6 (IL-6) is produced by bone and bone marrow-derived stromal cells and that it plays an important role in osteoclast development. Here we examined whether parathyroid hormone (PTH), calcitonin (CT), or the calcitonin gene-related peptide (CGRP) influence IL-6 production by two murine bone marrow-derived stromal cell lines: the preadipocyte-like stromal cell line +/+ LDA11 and the fibroendothelial stromal cell line MBA 13.2. We found that CGRP (but not PTH or CT) exerted a dose-dependent increase in cAMP and IL-6 production in the +/+ LDA11 cells. In addition, CGRP had an inhibiting effect on the proliferation of this stromal cell line. CGRP, however, did not affect cAMP or IL-6 in the rat osteogenic sarcoma cell line UMR-106-06, which exhibits CT receptors, whereas CT stimulated both cAMP and IL-6 by the UMR-106-01 cells. In contrast to the specificity of the IL-6 response of the +/+ LDA11 cells to CGRP, IL-6 production by the MBA 13.2 stromal cells was stimulated by PTH whereas CGRP or CT had no effect. These data suggest that bone marrow-derived stromal cells express receptors for either CGRP or PTH in a phenotype-specific manner and that, acting via these receptors, CGRP and PTH stimulate IL-6 production by stromal cells. In addition, the evidence for specific receptors for the neuropeptide CGRP in bone marrow stromal cells and an effect of CGRP on IL-6 raises the possibility for a role of cytokines in a putative interplay between neuronal stimuli and bone.
Prevalence of vertebral fracture in women and the relationship with bone density and symptoms: The chingford study
Authors: Dr. Spector Tim D. , Mccloskey Eugene V. , Doyle David V. , Kanis John A.
Published: February 2010

A population survey was performed to estimate the prevalence of vertebral fractures in women aged 45–69 and to determine their relationship to bone density and symptoms. Subjects were 1035 women aged 45–69 (mean 55.4 years, response rate 77%) from the age-sex register of a large 11,000-person general practice in Chingford, London. Thoracic and lumbar spine x-rays were read by a semiautomated quantitative method. Vertebral fractures were diagnosed using a variety of morphometric methods, including a new method we recently developed and the published methods of Melton and Eastell. These methods all detect abnormal ratios between anterior, central, or posterior vertebral height and between observed posterior vertebral height and values predicted from the posterior height of adjacent vertebrae. Bone mineral density (BMD) of lumbar spine L1–4 and neck of femur was measured by dual-energy x-ray absorptiometry (DXA). Using our method, 147, 14.2% (95% CI 12.0–16.2%) of the 1035 women, had minor fractures (at least two vertebral ratios 2–2.99 SD below the mean) and 20, 1.9% (95% CI 1.2–3.0%) of the total, had severe fractures (at least two ratios more than 3 SD below the mean). In the 147 women with minor fractures, bone density of the spine was not significantly lower than in the other 868 women, and reported back pain or loss of height was no more common. Women with multiple minor fractures did have lower bone density, by 0.4 SD. In the 20 women with severe fracture, bone density was significantly lower, by 0.6 SD. Loss of height was more common, but back pain was not. Using the method of Melton the prevalence of deformity was 10.2% and, for the Eastell 3 and 4 SD method, 9.7 and 1.3%, respectively, which is similar to published data from the Rochester population. Minor vertebral deformities are common in postmenopausal women, but they are not usually associated with pain, loss of height, or (unless multiple) reduced bone density. This suggests that they may not be of clinical or pathologic importance. Severe deformities associated with low bone density are rare in U.K. women under the age of 70.
Estrogen does not restore bone lost after ovariectomy in the rat
Authors: Abe T. , Chow J.W.M. , Lean J.M. , Chambers T.J.
Published: February 2010

We recently found that 17β-estradiol (E2) not only suppresses bone resorption but also stimulates bone formation in the cancellous bone of female rats. This raises the possibility that E2 treatment might restore the bone lost after ovariectomy in the rat. To test this, 13-week-old rats were ovariectomized (ox). After a further 13 weeks the animals were injected with E2 (4 mg or 40 μg/kg daily), human calcitonin (hCT) (3 IU/kg daily), (3-amino-1-hydroxypropylidene)-1-bisphosphonate (AHPrBP) (0.3 mg/kg twice per week), or a combination of E2 with hCT or AHPrBP, for 8 weeks. The bone volume at the tibial metaphysis of ox animals was approximately 40% of that of sham-operated controls at the end of the experiment. Although the bone volume of ox rats treated with E2 and/or hCT or AHPrBP was slightly higher than that of untreated ox rats, the increase was not significant. Neither E2 alone nor a combination of E2 with hCT or AHPrBP was associated with a higher bone volume than hCT or AHPrBP alone, suggesting no effect of E2 beyond that of inhibition of bone resorption. Histodynamic indices of bone formation were increased in untreated ox rats compared to controls but suppressed in E2-treated, hCT-treated, and AHPrBP-treated animals. These results emphasize the similar responses of rat and human bone, both of which not only show bone loss with estrogen deficiency, preventable by estrogen administration, but also show an inability of estrogen to restore bone lost as a result of estrogen deficiency.
Stable murine chondrogenic cell lines derived from c-fos-induced cartilage tumors
Authors: Dr. Wang Zhao-Qi , Grigoriadis Agamemnon E. , Wagner Erwin F.
Published: February 2010

This study describes the detailed characterization of four murine chondrogenic cell lines (wT2-1, wT2–7, wT2–8, and wT2–9) that were isolated from a cartilage tumor induced by the protooncogene c-fos in chimeric mice. All cell lines are clonal and display a fibroblastic morphology with a doubling time of 1–2 days. Northern blot analysis demonstrated that in addition to expressing high levels of exogenous c-fos, all clones express varying levels of the cartilage marker gene type II collagen in addition to type I collagen. The clones also expressed high levels of the AP-1 genes c-jun and fra-1. The doubling times of these clones did not change over a period of 14 months in culture. Most importantly, however, expression of type II collagen was maintained in all cell lines for 8 months in culture, and two cell lines maintained type II collagen expression when analyzed after 14 months. Interestingly, type I collagen expression was lost after long-term culture. Following injection into syngeneic and nude mice, all cell lines formed tumors containing areas with the morphologic appearance of hyaline cartilage, indicating that these cell lines are chondrogenic. Thus, these stable murine chondrogenic cell lines provide a useful tool for studying the transcriptional control of cartilage-specific gene expression, as well as the growth control of chondrogenic cells.
Effects of combined prostaglandin and alendronate treatment on the histomorphometry and biomechanical properties of bone in ovariectomized rats
Authors: Lauritzen D.B. , Balena R. , Shea M. , Seedor J.G. , Markatos A. , Le H.M. , Toolan B.C. , Myers E.R. , Rodan G.A. , Hayes W.C.
Published: February 2010

Prostaglandin E2 (PGE2) has been shown to stimulate both bone resorption and formation in experimental animals, leading to augmentation of trabecular and cortical bone. The amino bisphosphonate alendronate (ALN) is a potent inhibitor of bone resorption. The objectives of this study were to examine if PGE2 stimulation of bone formation was dependent on bone resorption and if the bone accrued as a result of PGE2 treatment contributed to bone strength. The 48 female Sprague-Dawley rats were assigned to six groups as follows: five groups (8/group) were ovariectomized at the age of 6 months. One group was sacrificed 2 months later to establish baseline conditions, and four groups were treated for 25 days with (1) vehicle, (2) PGE2 at 3 mg/kg/day, (3) ALN sc at 0.8 μg/kg/day, and (4) PGE2 + ALN at the respective doses. The sixth group served as nonovariectomized untreated controls. Histomorphometric analysis of 6–10 μm thick tibial sections after in vivo fluorochrome double labeling showed that treatment with PGE2 alone increased endo-cortical mineral apposition rate and bone formation rate, stimulated production of bone trabeculae in the marrow cavity, and increased cortical porosity. Combined ALN + PGE2 treatment prevented the resorption induced by PGE2 but not the stimulation of bone formation on endocortical and periosteal surfaces and resulted in a significant increase in cortical thickness. Consistent with these observations, the femoral midshaft tested to failure in three-point bending showed a significant increase in strength in the PGE2 + ALN group (181 ± 15 N) compared to time 0 controls (145 ± 23 N) or to the ovariectomized vehicle-treated group (141 ± 28 N). Similarly, there was a statistically significant increase in femoral midshaft stiffness. There was also a statistically significant increase in the ash content of bones from animals treated with PGE2 + ALN relative to vehicle-treated animals. No statistically significant differences among groups were observed in femoral neck or vertebral strength or stiffness. These findings show that PGE2 stimulation of bone formation is not blocked by inhibition of bone resorption and that combined treatment with PGE2 and the bone resorption inhibitor ALN increased the mechanical strength of ovariectomized rat femoral shaft compared to nonovariectomized animals.
Urinary hydroxypyridinium crosslinks of collagen as markers of bone resorption and estrogen efficacy in postmenopausal osteoporosis
Authors: Seibel M.J. , Cosman F. , Shen V. , Gordon S. , Dempster D.W. , Ratcliffe A. , Lindsay R.
Published: February 2010

Estrogen deficiency-induced bone loss has been associated with accelerated bone turnover. Levels of some biochemical markers, such as serum osteocalcin (BGP), tartrate-resistant acid phosphatase (TRAP), and urinary hydroxyproline (OHP), have been shown to be related to the rate of bone turnover. They may therefore be useful in identifying the individual at risk for osteoporosis and monitoring the efficacy of the treatment. Two recently discovered markers, urinary pyridinoline (PYD) and deoxypyridinoline (DPD), are apparently directly related to bone matrix degradation and may be more accurate markers of bone resorption than OHP or TRAP. To evaluate the effects of menopause, osteoporosis, and estrogen replacement on the excretion of these new markers, we measured the levels of PYD and DPD and other biochemical markers of bone turnover in four groups of women, premenopausal healthy (PRE), postmenopausal healthy (POST), postmenopausal osteoporotic (UTO), and postmenopausal osteoporotic with estrogen treatment (ETO). Significant increases in PYD, DPD, BGP, TRAP, and OHP were found in POST and UTO groups compared with PRE. These increases were blunted by estrogen treatment when the levels of each of the markers returned to PRE levels. When comparing POST and UTO groups, significant increases were observed in UTO only for PYD, DPD, and urinary calcium but not for OHP, BGP, or TRAP. With subgroups matched for age and years from menopause, only DPD discriminated between POST and UTO. Indices of bone formation co-varied with markers of bone resorption in the total population. Although this held true in PRE and ETO groups, the bone resorption markers were not well correlated with bone formation markers in POST or UTO groups when they were analyzed individually. None of the markers correlated with bone mineral density measurements. Our results suggest that (1) urinary pyridinium crosslinks are significantly elevated after menopause, and estrogen replacement reverses the increase in osteoporotic patients; (2) DPD may be the most discriminating marker of bone turnover; and (3) the lack of correlation between bone resorption and formation markers found in estrogen-deficient groups indicates the possibility of an imbalance in bone remodeling.
Effect of basic fibroblast growth factor on the growth and differentiation of adult stromal bone marrow cells: Enhanced development of mineralized bone-like tissue in culture
Authors: Pitaru Sandu , Kotev-Emeth Shlomo , Noff Dina , Kaffuler Shoshana , Savion Naphtali
Published: February 2010

Rat stromal bone marrow cells (SBMC) were shown to produce mineralized bone-like tissue in culture in the presence of dexamethasone, ascorbic acid, and β-glycerophosphate. The addition of 3 ng/ml of basic fibroblast growth factor (bFGF) resulted in a significant increase in formation of mineralized tissue. The present study was aimed at assessing the effect of bFGF on the proliferation and differentiation of SBMC and on the sequential development of mineralized bone-like tissue in culture. Transmission electron microscopy of bFGF-treated cultures demonstrated the development of a multilayered structure resembling mineralized bone tissue consisting of cell layers embedded within a heavy extracellular matrix. The matrix was rich in bundles of collagen fibers associated with extensive mineral deposits consisting of hydroxyapatite as determined by infrared spectrophotometry. The addition of 3 ng/ml of bFGF resulted in significant enhancement of [3H]thymidine and [3H]proline incorporation and protein accumulation by 12-, 2.5-, and 2.5-fold, respectively. bFGF treatment increased cAMP responsiveness, alkaline phosphatase activity, osteocalcin level, 45Ca2+ deposition, and mineralized-like tissue formation and induced the earlier expression of these markers in the treated culture. A biphasic sequence of events was observed during the development of mineralized bone-like tissue in bFGF-treated and control cultures. The first phase is characterized by cell proliferation and matrix accumulation and is reflected by a progressive increase in [3H]thymidine and [3H]proline incorporation until day 11. The second phase, which follows, is characterized by a sharp decline in cell proliferation and matrix accumulation and a concomitant expression of osteoblast differentiation as reflected by the progressive increase in alkaline phosphatase activity, mineral deposition, and osteocalcin expression. Treatment of cultures with bFGF accentuated this biphasic sequence of events. These results indicate that bFGF has the capacity to stimulate both the growth and the biochemical functions of SBMC obtained from a young adult animal.
New resorption assay with mouse osteoclast-like multinucleated cells formed in vitro
Authors: Tamura Tatsuya , Takahashi Naoyuki , Akatsu Takuhiko , Sasaki Takahisa , Udagawa Nobuyuki , Tanaka Sakae , Suda Tatsuo
Published: February 2010

We previously reported a procedure to obtain a preparation containing a large number of mouse osteoclast (OCL)-like multinucleated cells (MNCs) formed in cocultures of mouse osteoblastic and bone marrow cells in the presence of 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3]. The MNCs satisfied major criteria of OCLs, such as tartrate-resistant acid phosphatase (TRAP) activity, acid production, calcitonin (CT) receptors, and the ability to form resorption pits on bone slices. In this report, we describe a simple resorption assay system using MNC preparations. After culturing MNC preparations or disaggregated rat OCL preparations on dentin slices, they were stained with Mayer's hematoxylin. The stained area corresponded exactly with the resorption pits visualized by scanning electron microscopy and were measured using an image analysis system attached to a light microscope. Pit formation by MNCs was gradually enhanced by reducing the medium pH (pH 7.5 < 7.2 < 6.9). The plan area resorbed by MNCs increased linearly for up to 72 h. These results are very similar to those obtained with OCL preparations. In multiple standard assays with MNC preparations, more than 250 MNCs could be placed on a dentin slice, and the total area resorbed to a level of up to 9% of the whole surface within 48 h. In contrast, in multiple assays with OCL preparations, it was not easy to place more than 50 OCLs on a slice and the resorbed area was only 0.7% of the surface. The resorbing activity of the MNC preparation expressed by the resorbed area per a TRAP-positive MNC (4.4 ± 0.4 × 10−3 μm2) was greater than that of the OCL preparation (1.9 ± 0.4 × 10−3 μm2). Pit formation by MNCs was dose dependently inhibited by salmon and human CT (ED50: salmon CT, 10−14 M; human CT, 3 × 10−13 M). Bafilomycin A1, a specific inhibitor of vacuolar type H+-ATPase, also inhibited pit formation by MNCs at concentrations of 10−9 M and above. In contrast, estrogen at 10−12–10−6 M had no significant inhibitory effect on pit formation in this assay system. The assay system presented here is sensitive and reproducible and will be useful for examining the effects of various natural factors and synthetic compounds on osteoclastic bone resorption. We have previously established a culture system for examining MNC formation. Therefore, it is now possible to examine the effects of various test compounds on the recruitment of OCLs and pit formation by the same OCLs in a series of experiments.
Does weight-bearing exercise affect non-weight-bearing bone?
Authors: Tommerup Lorri J. , Raab Diane M. , Crenshaw Thomas D. , Smith Everett L.
Published: February 2010

It was previously reported that weight-bearing exercise increases femur periosteal formation in 3-year-old, 230 kg cross-bred sows. To determine if this weight-bearing exercise also stimulated non-weight-bearing bone, bone formation in the seventh rib from these same sows was measured histomorphometrically on the periosteal, endocortical, and osteonal envelopes. The sows were randomly assigned by body weight to basal (B, n = 6), control (C, n = 7), or trained (T, n = 7) groups. After 3 weeks of exercise adaptation, T walked on a treadmill for 17 weeks at 5 km/h, 20 minutes/day, 5 days/week, at 5% grade. Groups were sacrificed initially (B) or after 20 weeks (C and T). Periosteal mineral apposition rate (MAR) was calculated over 136 days. Osteonal and endocortical MAR were calculated over the 14 days prior to sacrifice. There were no effects of exercise on the periosteal or endocortical percentage double-labeled surface (dLS/BS), osteonal remodeling frequency (N.dL.On/B.Ar), or MAR in any bone envelope of the rib. In conclusion, weight-bearing exercise sufficient to stimulate periosteal formation in the femur did not activate formation in the rib. Bone response to weight-bearing exercise appears to be specific to the loaded bones.
In vivo effect of 1,25-dihydroxycholecalciferol on the proliferation and differentiation of avian chondrocytes
Authors: Dr. Farquharson Colin , Whitehead C.C. , Rennie J.S. , Loveridge N.
Published: February 2010

A combination of immunocytochemistry and in situ biochemistry has been used to determine the in vivo effects of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] on the proliferation and differentiation of chondrocytes. Chicks were fed a diet supplemented with 1,25-(OH)2D3 (2.5, 5, or 10 μg/kg diet) for 3 weeks, and measurements were made in sections of growth plate of chondrocyte proliferation and rate of maturation through the growth plate [using bromodeoxyuridine (BrdUrd) labeling] and also chondrocyte differentiation [assessed by alkaline phosphatase (ALP) activity]. The labeling indices of the control and supplemented chicks were similar (23.1 ± 1.3 versus 23.2 ± 1.6%); however, within a 21 h period the BrdUrd-positive cells of the supplemented chicks had moved down the growth plate significantly farther than in the control chicks (71.0 ± 2.8 versus 52.6 ± 1.8%). Greater ALP (mean integrated absorbance) activity higher up the growth plate of the supplemented chicks indicated a more differentiated phenotype in cells closer to the epiphyseal junction. Within individual transitional chondrocytes ALP activity in the 10 μg/kg supplemented chicks was 26.6 ± 0.85, which was significantly higher (p < 0.01) than that of the control chicks (19.2 ± 0.9). These results suggest that 1,25-(OH)2D3 in vivo does not increase the rate of chondrocyte prolferation but accelerates the onset of maturation.
Immunohistochemical detection and northern blot analysis of estrogen receptor in osteoblastic cells
Authors: Ikegami Akira , Inoue Satoshi , Hosoi Takayuki , Mizuno Yuzo , Nakamura Tetsuro , Ouchi Yasuyoshi , Orimo Hajime
Published: February 2010

Expression of estrogen receptor (ER) was studied in MC3T3-E1 cells (mouse osteoblastic cell line), HOS TE85 cells (human osteosarcoma cell line), and primary osteoblastic cells derived from mouse calvaria with immunohistochemical techniques. The staining of ER was readily detectable in MC3T3-E1 cells, HOS TE85 cells, and primary osteoblastic cells by using a monoclonal anti-ER antibody that recognizes the DNA binding domain of ER. The immunoreactivity was distributed in the cytoplasm as well as in the nuclei. 17β-Estradiol (10−8 M) did not alter this staining pattern. The expression of ER was confirmed by Northern blot analysis using rat ER cDNA probe, which revealed a 6.5 kb band in MC3T3-E1 cells and a 6.2 kb band in HOS TE85 cells. The mRNA level of ER was not altered by 17β-estradiol (10−8 M). The immunohistochemical studies showed that ER was not detectable in all cells but in a small population of each cell type. This study is the first report to demonstrate the presence of ER immunohistochemically, and our results suggest the heterogeneity of ER expression among osteoblastic cells.
Expression of annexin I, II, V, and VI by rat osteoblasts in primary culture: Stimulation of annexin I expression by dexamethasone
Authors: Suarez Françoise , Rothhut Bernard , Comera Christine , Touqui Lhousseine , Marie Françoise Russo , Silve Caroline
Published: February 2010

To determine whether rat osteoblasts synthesize proteins of the annexin family and to evaluate the extent to which glucocorticoids modulate the expression of annexins by these cells, osteoblasts were grown in primary cultures in the absence or presence of dexamethasone, and the expression of annexins was evaluated by immunoblotting using polyclonal antibodies against human annexins. Four different annexins (I, II, V, and VI) were found to be expressed by rat osteoblasts. The expression of annexin I, but not the other annexins studied, was increased in osteoblasts cultured in the presence of dexamethasone (173 ± 33% increase comparing untreated cells and cells treated for 10 days with 5 × 10−7 M dexamethasone). Increased expression of annexin I was observed after the third day of exposure to dexamethasone and rose thereafter until day 10; annexin I expression increased with dexamethasone concentrations above 10−10 M throughout the range of concentrations studied. The increase in annexin I protein was associated with an increase in annexin I mRNA and was completely blocked by the concomitant addition of the glucocorticoid receptor antagonist RU 38486. The increase in annexin I content following dexamethasone treatment was associated with an increase in alkaline phosphatase activity and PTH-induced cAMP stimulation, whereas phospholipase A2 activity in the culture medium was reduced to undetectable levels. The finding that four annexins are expressed in rat osteoblasts in primary culture raises the possibility that these proteins could play an important role in bone formation by virtue of their ability to bind calcium and phospholipids, serve as Ca2+ channels, interact with cytoskeletal elements, and/or regulate phospholipase A2 activity. In addition, the dexamethasone-induced increase in annexin I may represent a mechanism by which glucocorticoids modify osteoblast function.
Osteoblasts express the PMCA1b isoform of the plasma membrane Ca2+-ATPase
Authors: Meszaros J. Gary , Karin Norman J.
Published: February 2010

We report here that osteoblasts and osteoblast-like osteosarcoma cells express PMCA1b, an alternatively spliced transcript of plasma membrane Ca2+-ATPase. Synthetic oligonucleotide pairs were designed based upon unique regions of the cDNA encoding known PMCA isoforms (PMCA1–3) and used as primers in PCR-mediated amplification of cDNA synthesized from ROS 17/2.8 osteosarcoma cell RNA. A product was observed only when PMCA1-specific primers were present; no products were seen with PMCA2 or PMCA3 primers unless cDNA synthesized from rat brain RNA was present. Examination of the cDNA encoding the C terminus of PMCA1 from ROS 17/2.8 cells revealed that the mRNA is spliced to yield the PMCA1b isoform, a Ca2+-ATPase containing a consensus phosphorylation site for cAMP-dependent protein kinase A and a modified calmodulin binding domain. PMCA1b was also detected in UMR-106–01 osteosarcoma cells and unpassaged primary rat calvarial osteoblasts. These results suggest that the regulation of osteoblast function by agents that act via cAMP-mediated pathways may involve alterations in the activity of the plasma membrane Ca2+-ATPase.
Circulating levels of vitamins K1 and K2 decreased in elderly women with hip fracture
Authors: Hodges S.J. , Akesson K. , Vergnaud P. , Obrant K. , Prof. Delmas P.D.
Published: February 2010

We measured the serum levels of phylloquinone (vitamin K1) and of the menaquinones, MK-7 and MK-8, in a group of 51 women with a mean age of 81 years who were studied within a few hours after a hip fracture. A group of 38 healthy age-matched women randomly chosen from the same population served as controls. Patients with hip fracture had a marked reduction in serum vitamin K1 (336 ± 302 versus 585 ± 490 pg/ml, p < 0.01), MK-7 (120 ± 84 versus 226 ± 178 pg/ml, p < 0.001), and MK-8 (89 ± 113 versus 161 ± 145 pg/ml, p < 0.01), and a large number had undetectable levels, especially of MK-8. Vitamin K levels were not correlated with the time elapsed after fracture or with serum cortisol or other biochemical variables. These data suggest that patients with hip fracture have vitamin K deficiency, an abnormality that could affect bone metabolism through an impairment of the gamma carboxylation of the gla-containing proteins of bone.
Reactive oxygen species activate and tetracyclines inhibit rat osteoblast collagenase
Authors: Ramamurthy Nungavaram S. , Vernillo Anthony T. , Greenwald Robert A. , Lee H.-M. , Sorsa Timo , Golub Lorne M. , Rifkin Barry R.
Published: February 2010

Recent studies have demonstrated that tetracyclines (TCs) scavenge reactive oxygen species (ROS). Hypochlorous acid (HOCI), an ROS produced by neutrophils, has been shown to activate neutrophil procollagenase. The objective of the present study was to determine whether (1) HOCI also activated osteoblast procollagenase and (2) TCs inhibited this enzyme in the presence of HOCI. HOCI (5 μM) activated the proenzyme approximately sixfold (P < 0.01) from the medium of PTH-treated UMR-106–01 osteoblastic osteosarcoma cells as determined by functional collagenase assay (3H-methyl-labeled collagen substrate). Doxycycline (50–400 μM) and chemically modified tetracycline, CMT-1 (100–400 μM), significantly inhibited collagenase activity 50–90% and 40–80%, respectively, in the presence of 5 μM HOCI. Concentrations of 6–25 μM doxycycline and 10–50 μM CMT-1 had no significant effect. Furthermore, an excess concentration of cation (50 mM CaCI2 or 50 μM ZnCI2) added to the incubation mixtures containing either doxycycline or CMT-1 did not restore collagenase activity, as demonstrated by SDS-PAGE-fluorography. These data suggested that TCs reduced available HOCI and thus prevented the hypochlorous acid conversion of the osteoblast proenzyme to active collagenase. TCs may have therapeutic potential in the treatment of periodontitis and other diseases by several mechanisms that inhibit pathologic collagen breakdown.
Intestinal malabsorption of 45calcium in young Gy mice, a second model for X-linked hypophosphatemia
Authors: Woodward Jennifer E. , Meyer Martha H. , Gray Richard W. , Dr. Meyer Ralph A.
Published: February 2010

X-linked hypophosphatemia, a common metabolic bone disease in humans and mice (the Hyp and Gy mutations), is characterized by decreased plasma phosphate, decreased renal tubular reabsorption of phosphate, rickets, and osteomalacia. The question of whether intestinal malabsorption of calcium contributes to the bone disease is controversial. Intestinal absorption of 45Ca was studied in three different mouse colonies: Gy on B6C3H background, Hyp on B6C3H background, and Hyp on C57BL/6J background, all at 4 weeks of age. The duodenum was isolated by sutures, and 45Ca in a 150 mM NaCl and 2 mM CaCl2 solution at pH 7.2 was injected into the lumen. Absorption was measured by the amount of 45Ca remaining in the lumen and by the plasma isotope level. The Gy and Hyp mice of both sexes significantly malabsorbed 45Ca at 4 weeks of age compared to normal littermates. Following the 4 week study, intestinal absorption was measured at 2, 7–8, and 12 weeks of age in normal and Gy mice on the B6C3H background. At 2 and 7–8 weeks of age, the Gy males significantly malabsorbed 45Ca compared to their normal littermates. Serum 1,25-dihydroxyvitamin D was not significantly altered in Gy males at 4 weeks of age. This suggests the possibility of resistance of the intestine to stimulation. Malabsorption of calcium in young Gy and Hyp mice may exacerbate the low mineralization in their rachitic bone disease.
Effects of bone-seeking hormones on DNA synthesis, cyclic AMP level, and alkaline phosphatase activity in cultured cells from human posterior longitudinal ligament of the spine
Authors: Ishida Yoichiro , Kawai Shinya
Published: February 2010

In a study of the osteogenesis capability of the human posterior longitudinal ligament of the spine, ligament cells were isolated and cultured. The effect of bone-seeking hormones, such as parathyroid hormone (PTH), calcitonin (CT), prostaglandin E2 (PGE2), and 1,25-dihydroxycholecalciferol [1,25-(OH)2D3], on the ligament cells was investigated with respect to DNA synthesis, adenosine-3′,5′-cyclic monophosphate (cAMP) levels, alkaline phosphatase (ALP) activity, and acid phosphatase (ACP) activity. Cell lines obtained from nonossified sites in patients with ossification of the posterior longitudinal ligament of the spine (OPLL) were found to have several different phenotypic characteristics for osteoblasts: high ALP activity, PTH- and PGE2-stimulated increases in cAMP, and responses to both CT and 1,25-(OH)2D3. It is clear that proliferation and differentiation in such ligament cells are controlled by various types of bone-seeking hormones, and it was suggested that many cells with osteoblast-like characteristics are present. These results are considered important with respect to the etiology of OPLL, and an experimental system using cultured ligament cells appears to be useful in research on OPLL.