bluuebunny/crossref_metadata_2025 huggingface dataset:
Responsiveness of clonal limb bud cell lines to bone morphogenetic protein 2 reveals a sequential relationship between cartilage and bone cell phenotypes
Authors: Rosen Vicki , Nove John , Song Jeffrey J. , Thies R. Scott , Cox Karen , Wozney John M.
Published: February 2010

There is growing evidence to suggest that BMPs are among the signals necessary to create the embryonic skeleton, but how these regulatory molecules enter the pathways of embryonic bone formation remains to be defined. The earliest steps of endochondral bone formation, consisting of mesenchymal condensation and chondrogenesis, have been shown to result directly from BMP-2 action. To determine whether the transition from chondrogenesis to osteogenesis occurring later in endochondral bone formation is also the result of BMP activity, we tested the effects of BMP-2 on immortalized endochondral skeletal progenitor cells derived from mouse limb bud. The cell lines established by this process were found to fall into three general categories: undifferentiated skeletal progenitor cells, which in the presence of BMP-2 first express cartilage matrix proteins and then switch to production of bone matrix proteins; prechondroblast-like cells that constitutively express a subset of markers associated with chondrogenesis and, in the presence of BMP-2, shut off synthesis of these molecules and are induced to produce bone matrix molecules; and osteoblast-like cells that are not significantly affected by BMP-2 treatment. These data suggest that BMP-2 initiates the differentiation of limb bud cells into cells of both the cartilage and bone lineages in a sequential manner, making BMP-2 a potent regulator of skeletal cell differentiation.
Parathyroidectomy does not prevent bone loss in the oophorectomized rat
Authors: Sims N.A. , Dr. Morris H.A. , Moore R.J. , Durbridge T.C.
Published: February 2010

The evidence for a role of parathyroid hormone in the bone loss after the menopause remains controversial. This study examines the effect of parathyroidectomy on femoral trabecular bone volume, thickness, and spacing and biochemical markers of bone turnover in the oophorectomized rat. Female Sprague-Dawley rats 3 months old were double sham operated (sham), oophorectomized (OPX), parathyroidectomized (PTX), or oophorectomized and parathyroidectomized (O/P) under halothane anesthesia. At 9 weeks postoperation, femoral trabecular bone volume (BV/TV) was lower in OPX and O/P rats compared with sham or PTX animals (BV/TV, %, mean + SEM): sham 25.9 + 0.5, OPX 15.1 + 0.9, PTX 24.1 + 0.9, O/P 17.3 + 0.5; p < 0.001). Urinary hydroxyproline excretion, serum osteocalcin, and alkaline phosphatase activity were higher in OPX and O/P rats compared with control animals at 3 weeks postoperation (OHPE μM GF, mean + SEM: sham 1.37 + 0.16, OPX 2.16 + 0.26, PTX 0.95 + 0.21, O/P 1.92 + 0.22, p < 0.005; osteocalcin, μg/liter, sham 31.8 + 1.8, OPX 33.7 + 2.7, PTX 24.5 + 2.1, O/P 34.3 + 2.1, p < 0.025; alkaline phosphatase, U/liter, sham 90 + 3, OPX 125 + 9, PTX 87 + 9, O/P 116 + 11, p < 0.005). These data indicate postoophorectomy bone loss is not prevented by parathyroidectomy.
Structural requirements for bisphosphonate actions in vitro
Authors: van Beek Ermond , Hoekstra Monique , van De Ruit Marjan , Löwik Clemens , Papapoulos Socrates
Published: February 2010

We investigated the structural requirements for the binding of bisphosphonates to bone mineral and the relation between their affinity for bone and their effects on bone resorption in vitro. For this we used fetal mouse long bones in culture and bisphosphonates with variable R1 and R2 structures. In addition, we studied the effect of structural differences in the incorporation of calcium into bone. We found that bisphosphonates containing a hydroxyl group in the R1 position have the highest affinity for bone mineral. This was related to their capacity to inhibit the incorporation of calcium into long bones but not to their antiresorptive potency. The latter was primarily determined by R2. Furthermore, the effect of bisphosphonates on calcification, but not on resorption of bone explants, was mainly determined by the mode of addition. The continuous presence of bisphosphonate during culture inhibited calcification even at very low concentrations, but short incubation of the bones with relatively high concentrations had no effect. This is probably a result of differences in the availability of the compound to the process of calcification. Because, in vivo, the more potent bisphosphonates inhibit resorption without adversely affecting mineralization of the skeleton and they disappear rapidly from the circulation after administration, we suggest that cultures of bone explants incubated with bisphosphonates for short times rather than cultures in which the drugs are continuously present provide more accurate information about the in vivo effect of these compounds on both resorption and calcification.
Cholera toxin-stimulated bone resorption in cultured mouse calvarial bones not inhibited by calcitonin: A possible interaction at the stimulatory g protein
Authors: Dr. Ransjö Maria , Lerner Ulf H. , Ljunggren Östen
Published: February 2010

We examined the effect of calcitonin in cultured mouse calvarial bones after prestimulation with different activators of adenylyl cyclase. Calcitonin (100 ng/ml), added after 48 h of culture, inhibited bone resorption (assessed as release of 45Ca from prelabeled bones cultured for 96-144 h) stimulated with parathyroid hormone (PTH, 10 nM; 0-144 h) or the adenylyl cyclase stimulator forskolin (2 μM; 0-144 h). However, no effect of calcitonin was demonstrated when bone resorption was prestimulated with the adenylyl cyclase stimulator cholera toxin, at and above 1 ng/ml, at any time point studied. In contrast, two other types of inhibitors of bone resorption in vitro, the carbonic anhydrase inhibitor acetazolamide (10 μM) and the aminobisphosphonate AHPrBP (10 μM), significantly inhibited cholera toxin-stimulated bone resorption. No cyclic AMP response to calcitonin was seen after preculture for 48 h with cholera toxin (0.1-100 ng/ml), although bones precultured in basic medium, in the absence or presence of forskolin, were still able to respond to calcitonin with elevation of cyclic AMP. Binding studies with [125I]calcitonin demonstrated that the preculture with cholera toxin did not affect the binding of calcitonin to the receptor. In summary, our data show that cholera toxin pretreatment makes calvarial bones insensitive to calcitonin-induced inhibition of bone resorption as a result of an interaction with cholera toxin at the level of calcitonin receptor-linked signal transduction. We suggest that the interaction, distal to the calcitonin receptor, is caused by the irreversible activation of G s produced by cholera toxin. The study suggests that a functional G s coupled to adenylyl cyclase is required for transduction of the inhibitory action of calcitonin on bone resorption.
Femoral bone loss progresses with age: A longitudinal study in women over age 65
Authors: Greenspan Susan L. , Maitland Lauri A. , Myers Elizabeth R. , Krasnow Meryl B. , Kido Tamiko H.
Published: February 2010

Although previous longitudinal studies suggest bone loss ceases at the spine and radius in women over age 65, few data are available to determine whether femoral bone loss continues in this age group. Because a low femoral bone mass is associated with an increased risk of hip fracture and current therapies for fracture prevention focus on halting bone loss, it is imperative to determine whether femoral bone loss occurs in those who sustain 90% of hip fractures: women over age 65. To determine the annual rate of femoral bone loss, the association of bone loss and age, and the relationship between femoral and spinal bone loss in this age group, we prospectively followed femoral and spinal bone mineral density (BMD) in 85 healthy, community-dwelling, ambulatory women over age 65 (mean 77 years, range 66-93 years). Measurements of femoral and spinal BMD were assessed twice over 1 year using dual-energy x-ray absorptiometry. Cross-sectional analysis of site-specific baseline BMD suggested a significant bone loss in the femoral neck (-0.76% per year, p < 0.01), total hip (-0.70% per year, p < 0.01), trochanter (-0.71% per year, p < 0.05), intertrochanter (-0.88% per year, p < 0.01), and Ward's triangle (-0.86% per year, p < 0.01) but no significant change at the spine. Longitudinal analysis (mean + SD) revealed a similar loss of bone at the total hip (-0.95 + 2.88% per year, p < 0.01), trochanter (-0.74 + 3.62% per year, p < 0.01), and intertrochanter (-1.14 + 3.54% per year, p < 0.01), with a significant gain in lumbar BMD (0.94 + 3.34% per year, p < 0.05). The annual percentage change in BMD at the total hip was negatively correlated with age (p < 0.05). These data demonstrate that femoral bone loss is present in women over age 65 and that measurements of spinal BMD may be a misleading reflection of femoral skeletal integrity in this age group. Furthermore, these data provide the scientific rationale to develop and implement femoral bone loss prevention strategies in this important target group.
Bone-forming ability of 24r,25-dihydroxyvitamin d3 in the hypophosphatemic mouse
Authors: Yamate Tomoo , Tanaka Hiroyuki , Nagai Yumiko , Yamato Hideyuki , Taniguchi Nobuyuki , Nakamura Toshitaka , Seino Yoshiki
Published: February 2010

To determine whether 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] exerts unique biologic effects on bone, we examined the effects of the vitamin D metabolites, 24R,25(OH)2D3 and 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], on the hypophosphatemic (Hyp) mouse, a model for X-linked hypophosphatemic rickets in humans. The Hyp mice were administered 1-10,000 μg/kg/day of 24R,25(OH)2D3, 0.01-10 (μg/kg/day of 1α,25(OH)2D3, or vehicle alone, given daily for 28 days by intraperitoneal injection. 24R,25(OH)2D3 at doses of 1-1000 μg/kg/day had dose-dependent effects in increasing bone size, dry bone weight, and bone mineral content without causing hypercalcemia. 1α,25(OH)2D3 at doses of 1 or 10 μg/kg/day, which we considered to have activity similar to that of 1000 μg/kg/day of 24R,25(OH)2D3 with respect to cell differentiation activity, caused severe bone resorption and hypercalcemia. At 0.1 μg/kg/day, 1α,25(OH)2D3 increased bone size, similarly to a dose of 1000 μg/kg/day of 24R,25(OH)2D3, without significantly affecting dry bone weight or bone mineral content, as did 1000 μg/kg/day of 24R,25(OH)2D3. These findings suggest that 24R,25(OH)2D3 exerts unique activity in the Hyp mouse rather than merely mimicking the activity of 1α,25(OH)2D3.
Premenopausal and postmenopausal changes in bone mineral density of the proximal femur measured by dual-energy x-ray absorptiometry
Authors: Ravn Pernille , Hetland Merete Lund , Overgaard Kirsten , Christiansen Claus
Published: February 2010

Total and regional bone mineral density (BMD) of the proximal femur was measured by DXA in 1238 healthy white women. In the 389 premenopausal women, aged 21-54 years, no bone loss was observed before the menopause, except in the femoral neck and Ward's triangle, in which BMD decreased by 0.3%/year (SEE 0.2-0.9%/year, p < 0.001) and 0.6%/year (SEE 0.4-0.8%/year, p < 0.001), respectively. In the postmenopausal women aged 48-75 years, there was a highly significant exponential decay in BMD with age and years since menopause (YSM) in all regions (-0.58 < r < -0.48, p < 0.001). However, YSM was a better predictor of BMD than age. The decrease in BMD in the First 5 years postmenopause reached values of 9-13%. The estimated bone loss after 20 years was 17-30%, greatest in Ward's triangle and smallest in the intertrochanteric region. BMD correlated highly significantly with BMI (0.26 < r < 0.48, p < 0.001). In conclusion, the present study indicates a stable premenopausal bone mass of the proximal femur and a postmenopausal bone loss, which is influenced mainly by YSM within the first 10-15 years after menopause. BMD correlated with body mass index (BMI) in the postmenopausal years, confirming that low BMI constitutes a potential risk factor for osteoporosis.
Direct demonstration that the vitamin k-dependent bone gla protein is incompletely γ-carboxylated in humans
Authors: Cairns James R. , Dr. Price Paul A.
Published: February 2010

Incomplete vitamin K-dependent γ-carboxylation has been found in bone Gla protein (BGP) isolated from each of 20 different human bone samples. Using N-terminal protein sequencing of the methyl-esterified protein (Anal Biochem 1991;199:93-97), a method that directly measures the percentage of γ-carboxylation at each target glutamate residue, the extent of incomplete BGP γ-carboxylation was found to depend strongly on sequence position, with (x̄ + SD) 67 + 14% γ-carboxylation at residue 17, 88 + 9% γ-carboxylation at residue 21, and 93 + 4% γ-carboxylation at residue 24. There is a strong correlation between the incomplete γ-carboxylation at glutamate residues 17 and 21 for BGP purified from the 20 bone samples (p < 0.001), which suggests that individual differences in the efficiency of BGP γ-carboxylation during synthesis probably cause the observed differences in percentage BGP γ-carboxylation between bone samples. These results have been interpreted using a kinetic treatment of γ-carboxylation. This treatment predicts the existence of differences in the extent of γ-carboxylation between glutamate residues in BGP, as well as the correlation between percentage carboxylation at Glu17 and Glu21. Although the molecular basis of incomplete BGP γ-carboxylation is at present unknown, if incomplete BGP γ-carboxylation were caused only by differences in the availability of vitamin K in bone cells, this kinetic treatment predicts that the range in BGP γ-carboxylation observed in the 20 human bone samples studied here could be explained by a relatively modest fivefold range in the vitamin K levels of these individuals.
Effects of passive immunization against parathyroid hormone-related protein: PTHrP is the responsible factor in mediating hypercalcemia in the walker carcinosarcoma 256 rat model
Authors: Schilling Tobias , Blind Eberhard , Baier Reinhard , Sinn Hans-Peter , Moallem Eli , Silver Justin , Ziegler Reinhard , Raue Friedhelm
Published: February 2010

The Walker carcinosarcoma (WCS) 256 is a well-characterized rat model of humoral hypercalcemia of malignancy (HHM). We addressed the question of whether parathyroid hormone-related protein (PTHrP) is the factor responsible for mediating HHM in this model. WCS 256 cells were subcutaneously implanted in female rats. We examined the plasma at days 0,2,4,6, and 8. The midregional PTHrP measured by radioimmunoassay (RIA) and the plasma calcium increased significantly. Measuring PTHrP by a two-site immunoradiometric assay (IRMA) showed comparable results. There was a strong positive correlation between plasma calcium and midregional PTHrP (r = 0.85, p < 0.0001). A strong positive correlation between tumor weight and both midregional PTHrP (r = 0.83, p < 0.0001) and plasma calcium (r = 0.87, p < 0.0001) was also found. After surgical removal of the tumor at day 5, both plasma calcium and plasma PTHrP levels fell to within the normal range. Ip administration of native polyclonal antiserum against PTHrP(53–84) led to a significant decrease of plasma calcium. Extracted WCS 256 tumor showed 5-fold increased levels of midregional PTHrP compared with liver. Immunohistochemistry and Western blot were positive for PTHrP. RNA from the WCS 256 tumor was positive for PTHrP whereas liver tissue RNA was negative. WCS 256 cells grown in vitro also secreted PTHrP into the medium. We conclude that PTHrP is synthesized and secreted by WCS 256 and that PTHrP is the factor responsible for mediating hypercalcemia in the WCS 256 rat model.
Prostaglandin E2 administration prevents bone loss induced by orchidectomy in rats
Authors: Li M. , Jee Webster S. S. , Ke H.Z. , Tang L.Y. , Ma Y.F. , Liang X.G. , Setterberg R.B.
Published: February 2010

The objects of this study were to investigate whether prostaglandin E2 (PGE2) can prevent orchidectomy (ORX)-induced cancellous bone loss in growing male rats, and to determine the differential effects of PGE2 on sham-operated (sham) and ORX male rats. Fourteen-week-old Sprague-Dawley male rats were divided into groups of basal, vehicle-treated sham, PGE2-treated sham, vehicle-treated ORX, and PGE2-treated ORX rats for either 3 or 9 weeks. PGE2 was given at 6 mg/kg body weight daily by subcutaneous injection. Static and dynamic cancellous bone histomorphometry were performed on double-fluorescent labeled undecalcified proximal tibial metaphyseal sections. No effect was observed by ORX on body weight or longitudinal bone growth rate when compared with sham-operated controls. However, androgen deficiency caused significant increases in percent eroded perimeter, mineral apposition rate, and bone turnover (bone-volume-referent-bone formation rate), which resulted in a significant decrease in trabecular bone number, increase in trabecular separation, and a nonsignificant decrease in trabecular bone area by 3 weeks of ORX. After 9 weeks of ORX, trabecular bone area and number were significantly decreased, and trabecular separation, percent eroded perimeter, and the index of bone turnover (bone-volume-referent-bone formation rate) remained significantly increased while the index of bone formation (tissue-volume-referent-bone formation rate) was nonsignificantly decreased when compared with sham controls. When 6 mg PGE2kg/day was given for 3 and 9 weeks, similar anabolic effects were observed in sham and ORX rats. PGE2 caused significant decreases in body weight and longitudinal bone growth rate and significant increases in trabecular bone area, thickness, labeling perimeter, mineral apposition rate, and tissue-volume-referent-bone formation rate in both sham and ORX rats when compared with their respective controls. In sham-operated rats, PGE2 had no effect on percent eroded perimeter after 3 weeks of treatment, whereas after 9 weeks PGE2 caused a significant increase in this index. PGE2 partially inhibited the increase in percent eroded perimeter induced by ORX at week 3, but had no effect on this parameter at week 9 as compared with ORX controls. In summary, the new findings from current study indicated that PGE2 can prevent bone loss induced by ORX and the anabolic skeletal effect of PGE2 independent of the presence of androgen and longitudinal growth and occurs mainly on the pre-existing bone surface.
Interleukin-1 modulates phosphorylation of proteins in human osteoblastic cells
Authors: Kang Yung-Ming , Yeh Yu-Ling , Dr. Graves Dana T.
Published: February 2010

Interleukin-1 (IL-1) is a potent bone resorbing cytokine with diverse biological effects. We previously reported that IL-1 inhibits PDGF-AA-induced biological activities including PDGF-AA-induced tyrosyl phosphorylation. In the present studies, we first investigated and compared the tyrosyl phosphorylation pattern induced by EGF, IGF-1, PDGF-AA, and bFGF in human osteoblastic cells. We then examined the effect of IL-1 on the tyrosyl phosphoproteins induced by each ligand. Immunoblot analyses show that EGF, IGF-1, and PDGF-AA each elicit a different pattern of tyrosyl phosphorylated proteins in normal human osteoblastic cells. IL-1β inhibits PDGF-AA induced autophosphorylation by down-regulation of the PDGF-α receptor, as demonstrated by immunoprecipitation experiments. For other ligand-induced tyrosyl phosphoproteins, IL-1β reduced the intensity of EGF-induced pp55,000, and IGF-1 induced pp185,000 and pp 175,000. These experiments indicate that IL-1 inhibits phosphorylation of specific proteins induced by growth factors. By using inhibitors of secondary message pathways, we determined that the inhibitory effect of IL-1β on PDGF-AA receptor binding and receptor tyrosyl autophosphorylation was not dependent on protein kinase A, protein kinase C, or the formation of prostaglandins. These data suggest the existence of an alternative pathway that may participate in IL-1β signaling.
Cyclical treatment of osteopenic ovariectomized adult rats with PTH(1–34) and pamidronate
Authors: Cheng P.-T. , Chan C. , Müller K.
Published: February 2010

Administration of PTH(1–34) at low intermittent doses stimulates bone formation in vivo. However, the new bone is quickly resorbed after drug withdrawal.(24) Following the concept of lose, restore, and maintain,(27) we studied the possibility of maintaining the PTH(1–34)-mediated new bone by APD post-treatment and the effect of a second PTH(1–34)-APD cycle in osteopenic ovariectomized (OVX) adult rats. Eighty OVX rats (6-months-old, 3 months after OVX) and 10 sham-operated rats were divided into nine groups. One OVX group was killed as baseline (BL). Five OVX groups were injected with PTH(1–34) (20 μg/kg/day, subcutaneously) for 3 weeks (5 days/week). The remaining two OVX groups and the sham group (S) were injected with saline as controls. One PTH(1–34) group (A) and one control group (B) were then killed. Three PTH(1–34) groups were post-treated with APD injections (250 μg/kg/day, subcutaneously) for 5 days; the remaining PTH(1–34) group, the other control group, and group S were injected with saline; and all these groups were left untreated for 3 weeks. At the end, one PTH-APD group (C), the PTH-saline group (D), and the saline-saline group (E) were killed. Another PTH-APD cycle was applied to the remaining two PTH-APD groups, with group F killed after the second PTH treatment and group G killed after two complete cycles. We found a 90% increase in cancellous bone volume (Cn.BV/TV) and a 28% increase in trabecular thickness (Tr.Th) in group A over group B. Group C had a 78% increase in Cn.BV/TV and a 64% increase in Tr.Th over group E, but groups D and E were not different. Group G had another 15% increase in Cn.BV/TV over group C, but groups F and C were not significantly different. The results showed that APD post-treatment did maintain the PTH(1–34)-mediated new bone and that the effect of a second PTH(1–34)-APD cycle was minor, probably due to the prolonged inhibitory effect of APD on bone formation.
Effect of growth hormone on bone: Bone mineral density, trabecular bone volume, and alkaline phosphatase improve or are restored in the dwarf rat treated with growth hormone
Authors: Wright Nancy M. , Renault Josette , Hollis Bruce , Bell Norman H. , Key Lyndon L.
Published: February 2010

Studies were performed to determine whether serum total alkaline phosphatase (SAP), an index of bone formation; body weight; total body bone mineral density (BMD), measured by dual-energy X-ray absorptiometry; and tibial trabecular bone volume (TBV), measured by histomorphometry, are reduced in 2-week-old female sexually immature Lewis dwarf (dw/dw) rats (DW-CT, n = 9) with isolated growth hormone (GH) deficiency and, if so, whether recombinant human GH (rhGH), 200 μg/day subcutaneously for 4 weeks (DW-GH, n = 7), restores them. Studies were also performed to determine if 30% dietary restriction in 2-week-old female Lewis rats (LW-DR, n = 11) alters SAP, body weight, total BMD, or TBV compared with pair-fed controls (LW-CT, n = 7) given an ad libitum diet. Mean SAP (91 ± 5 versus 109 ± 5 U/l), body weight (102 ± 11 versus 140 ± 10 g), total BMD (88.5 ± 03 versus 101.4 ± 2.0 mg/cm2), and TBV (19.0 ± 1.0 versus 27.0 ± 1.4%) were significantly lower in DW-CT than in LW-CT animals, p < 0.05. In DW-GH, rhGH significantly increased mean SAP (130 ± 7 U/l), body weight (133 ± 10 g), total BMD (92.7 ± 13), and TBV (24.0 ± 1.9) compared with DW-CT animals. Compared with LW-CT rats, mean body weight and TBV were not different, but mean SAP was significantly higher (p < 0.01) and mean total BMD was significantly lower (p < 0.003) in DW-GH rats. In LW-DR compared with LW-CT animals, dietary restriction significantly reduced mean body weight (p < 0.05) but did not alter mean SAP, total BMD, or TBV. Thus, body weight, SAP, total BMD, and TBV were lower in DW-CT than in LW-CT rats. In the dose administered, SAP is markedly increased and reductions in body weight, total BMD, and TBV are completely or partially restored by treatment with rhGH in young female rats caused by isolated GH deficiency. Dietary restriction in young female rats with intact secretion of GH prevents weight gain but does not alter SAP, total BMD, or TBV compared with animals fed ad libitum.
The effects of risedronate on canine cancellous bone remodeling: Three-dimensional kinetic reconstruction of the remodeling site
Authors: Boyce R.W. , Paddock C.L. , Gleason J.R. , Sletsema W.K. , Eriksen E.F.
Published: February 2010

To investigate the dose-dependent effects of risedronate on cancellous bone remodeling, adult female beagle dogs were treated with either placebo, 0.1, 0.5, or 2.5 mg/kg/day of risedronate orally in an intermittent cyclic regimen (7 days on 21 days off), repeated three times. Iliac cancellous bone samples were subjected to histomorphometric analysis and three-dimensional (3-D) kinetic reconstruction of the remodeling site was performed. In the 0.1 mg/kg dose group, resorption and activation indices were no different from the placebo group. However, wall thickness was increased resulting in a positive bone balance at the level of the remodeling unit. In the 0.5 and 2.5 mg/kg dose groups, a dose-dependent reduction in activation frequency and tissue level bone formation was observed. Resorption rates were also significantly decreased, 60% and 80% for the 0.5- and 2.5-mg/kg groups, respectively. An approximate 25% reduction in final erosion depth was noted in both these groups. Analyses of the growth curves of the bone packet confirmed that the kinetics of the growth of a completed packet were different in the 0.5- and 2.5-mg/kg dose groups compared with placebo. These changes were associated with a significant increase in the final wall thickness in both groups indicating no net impairment of osteoblast function. These increases in wall thickness in combination with the reductions in final erosion depth in the 0.5 and 2.5 mg/kg groups resulted in a significant dose-dependent positive bone balance. This pharmacological profile suggests that risedronate may be of therapeutic utility in the treatment of metabolic bone diseases where reductions in activation frequency and resorptive cell activity at the level of the remodeling unit are a therapeutic goal.
Decreased bone density in inflammatory bowel disease is related to corticosteroid use and not disease diagnosis
Authors: Bernstein Charles N. , Seeger Leanne L. , Sayre James W. , Anton Peter A. , Artinian Lucy , Shanahan Fergus
Published: February 2010

Although corticosteroid therapy is associated with the development of osteopenia, it is unclear whether the cause of osteopenia in inflammatory bowel disease (Crohn's disease and ulcerative colitis) is related to corticosteroid therapy or other disease-related variables. Patients with Crohn's disease (a diffuse gastrointestinal disease) could have greater osteopenia than patients with ulcerative colitis because of small bowel disease and secondary malabsorption of calcium and vitamin D. A cross-sectional analysis of consecutive patients with Crohn's disease and ulcerative colitis was undertaken. Bone density was determined by measurements of the L2–L4 spine, the total hip, and Ward's triangle using dual energy X-ray absorptiometry (DXA). A number of clinical parameters were recorded prior to bone density evaluation and analyzed by univariate and subsequently multivariate analysis to determine possible predictors of osteopenia. Of the 26 patients with Crohn's disease, diminished bone density (a Z score of at least −1) was found at the hip in 64% and at the spine in 44%; and of the 23 patients with ulcerative colitis diminished bone density was found at the hip in 43% and at the spine in 48%. Among all the variables tested, only corticosteroid use was a statistically significant predictor of diminished bone density (p = 0.025 for the spine and hip and p = 0.005 for Ward's triangle). Disease diagnosis (Crohn's disease compared with ulcerative colitis) did not predict or correlate with diminished bone density. No obvious associations were seen between the measurements of any serum hormones or biochemistries and bone density, although the patients using corticosteroids had lower serum calcium levels than the nonusers. Separate multivariate analyses were performed for males and females. Corticosteroid use was statistically significantly associated with diminished bone density in females but not in males. All patients with inflammatory bowel disease (both Crohn's disease and ulcerative colitis), independent of whether or not they have small bowel disease, who have been using corticosteroids for long periods should have their bone density status investigated, since they have a high prevalence of diminished bone density and, therefore, are at risk for bone fractures. Further studies are required to sort out factors that may make bone density in females more sensitive to the effects of corticosteroids than that of males.
Prediction of fracture risk using axial bone mineral density in a perimenopausal population: A prospective study
Authors: Kröger Heikki , Huopio Jukka , Honkanen Risto , Tuppurainen Marjo , Puntila Eero , Alhava Esko , Saarikoski Seppo
Published: February 2010

Several prospective studies have shown that the bone mineral density (BMD) measured in the appendicular or axial skeleton has an inverse relationship with the risk of subsequent fractures. However, most of these studies have concentrated on relatively old age groups, and the usefulness of measuring BMD at the time of menopause has not been established. In the present study, BMD was measured at the lumbar spine and femoral neck by dual X-ray absorptiometry (DXA) in a random stratified population sample of 3222 perimenopausal women (mean age 53.4 years, range 47–59 years). These women were followed for fractures over a period of 2 years. The fractures reported by a postal inquiry were verified from medical records. Fractures sustained in motor vehicle accidents were excluded from the analyses. During a mean follow-up of 2.4 years, 183 fractures occurred in 168 women. Wrist (n = 47), ankle (n = 31), and rib (n = 28) were the most common sites of a fracture. Women in the lowest quartile of spinal BMD had a 2.9 times greater risk of fracture than those in the highest quartile. The respective risk increased 2.2 times from the lowest to the highest quartile of femoral BMD, respectively. The relative risk for suffering from any fracture per one SD decrease in BMD was 1.50 (95% CI; 1.27–1.76) for the spine and 1.41 (1.21–1.64) for the femoral neck. The present study demonstrates that bone mass is important in the pathogenesis of fractures even in perimenopausal women. We conclude that the axial BMD measurement at the time of menopause can be of use in predicting subsequent fracture risk.
Dual X-ray absorptiometry of the proximal femur: Normal european values standardized with the european spine phantom
Authors: Pearson J. , Dequeker J. , DR. Reeve J. , Felsenberg D. , Henley M. , Bright J. , Lunt M. , Adams J. , Curiel Diaz M. , Galan F. , Geusens P. , Jaeger P. , Kröger H. , Lips P. , Mitchell A. , Cano Perez R. , Pols H. , Rapado A. , Reid D.M. , Ribot C. , Schneider P. , Laval-Jeantet A.M. , Ruegsegger P. , Kalender W.
Published: February 2010

Measurements of the proximal femur by dual X-ray absorptiometry (DXA) are assuming increasing importance in clinical and epidemiological studies. However, different DXA manufacturers have adopted varying approaches to measuring the femoral neck and trochanter regions with the result that there is as yet no agreement on normal ranges and how to cross-calibrate between different machines. In this Concerted Action (of the EU's 2nd Framework Programme), 12 clinical centers in eight countries recruited a total of 855 female and 517 male Caucasian subjects who were judged to be free of conditions known to cause secondary osteoporosis and who had not been treated with drugs known to affect bone mass. The DXA machines used were made by Hologic, Lunar, and Norland. Each machine was cross-calibrated with the European Spine Phantom prototype designed by Kalender. All femoral neck and trochanter results were expressed as standardized values and were first examined for possible bias due to between-brand differences in choice of areas for measurement. On average, trochanter areas were similar between brands but Norland chose a narrower neck region for analysis. Bone mineral density (BMA or BMD g/cm) was log-normally distributed for all groups at the femoral neck but normally distributed at the trochanter in men and postmenopausal women. There were substantial between-center differences after cross-calibration in age-adjusted mean values at both sites and also in the rates of apparent bone loss with age and the amounts of scatter within populations about mean age-adjusted values. With the proviso that locally derived normal ranges would allow more accurate comparisons for clinical purposes, European normal ranges were derived for both neck and trochanter. Data obtained with the three individual brands of machine fitted these overall ranges well. Bone loss was apparent in premenopausal women at the femoral neck but not the trochanter. Postmenopausal women's data was somewhat better fitted by reference to years since menopause than to chronological age. This work provides a basis for conducting future epidemiological and clinical studies with more than one brand of machine as long as each individual subject, if measured consecutively, is measured on the same machine throughout.
The matrix Gla protein gene is a marker of the chondrogenesis cell lineage during mouse development
Authors: Luo Guangbin , D'Souza Rena , Hogue Deborah , DR. Karsenty Gerard
Published: February 2010

Matrix Gla protein (MGP) is, along with osteocalcin, a skeletal member of the family of extracellular mineral-binding Gla proteins. Although the precise function of these proteins remains obscure, circumstantial evidence suggests that they play a role in endochondral ossification. As a first step toward understanding MGP function we have performed a preliminary characterization of its promoter element and studied the developmental pattern of expression of this gene. DNA transfection experiments indicate that the mouse MGP promoter functions better in cells expressing the MGP gene than in cells that do not express the gene. During mouse development, MGP gene expression is detectable as early as day 10.5 of embryonic development (E10.5), before any skeletal structures are identifiable. In situ hybridization analysis shows that MGP mRNA is initially present at the mesenchymal epithelial interphase in lung and limb buds. As development proceeds, MGP gene is predominantly expressed in cells of the chondrocytic lineage in areas that will undergo endochondral ossification as well as in areas that will remain cartilaginous, such as the trachea and bronchi. In growth plate cartilage, MGP mRNA is present in resting, proliferative, and late hypertrophic chondrocytes. Surprisingly, MGP mRNA is absent from the early hypertrophic chondrocytes and from the osteoblasts. Finally, the MGP gene is expressed at a lower level in kidney medulla and uterus smooth muscle but not in brain, spleen, or heart during development. This study demonstrates that during development MGP gene expression occurs early and is predominant at the epithelial mesenchymal interfaces, principally of lung and limb buds, and in cells of the chondrocytic lineage. This finding raises the intriguing possibility that MGP may play distinct roles during embryogenesis and in the adult organism.
Ultrasound velocity through bone predicts incident vertebral deformity
Authors: Heaney R.P. , Avioli L.V. , Chesnut C.H. , Lappe J. , Recker R.R. , Brandenburger G.H.
Published: February 2010

We followed 130 postmenopausal women without evidence of vertebral deformity by lateral spine radiographs on entry into study for 2 years, and repeat spine radiographs were taken at the end of that time. Incident deformities occurring within this 2 year period were detected by two methods, a level-specific radiogrammetric approach and visual inspection by skilled clinicians. Fourteen incident deformities were detected by the radiogrammetric method, and 19 by the clinicians. Ultrasound transmission velocity was measured at the patella in each subject on entry. Values for ultrasound velocity were significantly correlated with incident fracture occurrence, with individuals having velocity values more than one standard deviation below the mean for the group exhibiting from 3.3 to 4.6 times the probability of incident fracture as individuals with velocity values more than one standard deviation above the mean. Thus, low values for ultrasound transmission velocity at the patella detect yet-unexpressed bony fragility at the spine and predict future fracture.
Sources of interracial variation in bone mineral density
Authors: Dr. Cundy Tim , Cornish Jill , Evans Margaret C. , Gamble Greg , Stapleton Joanne , Reid Ian R.
Published: February 2010

Many studies have demonstrated significant differences in bone mineral density between various racial groups. Although it has been suggested that differences in body weight contribute to such interracial variation, the artifactual effect of the skeletal size inherent in projectional absorptiometry methods has been largely ignored. We have measured bone mineral density by dual-energy X-ray absorptiometry in the lumbar spine and at three femoral sites in 200 premenopausal women of Chinese, Indian, European, or Polynesian origin (50 of similar mean age in each group). In the Chinese and Indian women the measured bone mineral density measurements (g/cm) were similar, but significantly less, at all sites, than those of European women (p ≤ 0.005). The European women were, however, significantly taller than both the Chinese and Indian women (p < 0.0001), and when the scale artifact of absorptiometry was removed by dividing the measured bone mineral density either by the height of the subject, or by the square root of the area over which the X-ray beam was projected, then the differences in mean bone mineral density between the Chinese, Indian, and European women were almost completely eliminated. The Polynesian women were significantly more obese (as judged from mean body mass index) than all the other groups (p < 0.0001) and had significantly greater bone mineral density at all sites than all the other groups both before (p < 0.0001) and after (p < 0.0001) correcting for the scale artifact. Analysis of covariance suggested that 10–40% of the remaining increase in bone density could be accounted for by their greater obesity, the balance representing a true interracial difference. This effect was most obvious in the lumbar spine and least evident at the femoral sites. We conclude that there is a true interracial difference in bone mineral density between Polynesians and the other races studied, and that this is increased further by the greater body weight of Polynesians. The differences between Europeans and Asians are substantially attributable to a measurement artifact arising from the smaller skeletal dimensions of Asians. This needs to be borne in mind by clinicians assessing bone density in Asian patients.
Rat tail suspension reduces messenger RNA level for growth factors and osteopontin and decreases the osteoblastic differentiation of bone marrow stromal cells
Authors: Zhang Renwen , Supowit Scott C. , Klein Gordon L. , Lu Zhaofeng , Christensen Monique D. , Lozano Renaud , Dr. Simmons David J.
Published: February 2010

We previously reported that bone marrow stromal cells produce insulin-like growth factors (IGF-I and -II), and that medium conditioned by marrow stromal cells stimulates osteoblast proliferation in vitro. The present study employed the rat tail-suspension model to unload the hindlimbs. It was designed to test the hypothesis that the development of osteopenia or osteoporosis could be due to a deficit in the osteogenic function of marrow stromal cells. Although tail suspension suppressed body weight during the first 3 days of an 11-day pair-fed study, the overall weight gain recorded by these animals was normal. Nevertheless, bone growth was inhibited by suspension. Similarly, the total adherent marrow stromal cell population harvested from the femurs and tibias was decreased by tail suspension, and only half the normal number of fibroblastic stromal cell colonies grew when they were cultured. The proliferation of alkaline-phosphatase-positive cells in the stroma was also inhibited. Northern hybridization revealed that the messenger RNA level for transforming growth factor-β2 and IGF-II in stromal cell was reduced by tail suspension. The production of IGF-II by marrow stromal cells was also decreased. The steady-state level of five different transcript sizes of IGF-I mRNA was altered differentially by tail suspension. Osteopontin mRNA was also reduced in marrow stromal cells from tail-suspended rats compared with the normal rats. These data suggest that skeletal unloading not only alters the mRNA level for growth factors and peptide production, but also affects the proliferation and osteogenic differentiation of marrow stromal cells. These changes may be responsible for the reduced bone formation in osteopenia and osteoporosis.
Differential effects of swimming versus weight-bearing activity on bone mineral status of eumenorrheic athletes
Authors: Taaffe Dennis R. , Snow-Harter Christine , Connolly Declan A. , Robinson Tracey L. , Brown Monte D. , Marcus Robert
Published: February 2010

To examine the role of skeletal loading patterns on bone mineral density (BMD), we compared eumenorrheic athletes who chronically trained by opposite forms of skeletal loading, intensive weight-bearing activity (gymnastics, n = 13), and nonweightbearing activity (swimming, n = 26) and 19 nonathletic controls. BMD (g/cm) of the lumbar spine, femoral neck, trochanter, and whole body was assessed by dual energy X-ray absorptiometry (DXA). Subregion analysis of the whole body scan permitted BMD evaluation of diverse regions. Swimmers were taller (p = 0.0001), heavier (p > 0.005), and had a greater bone-free lean mass (p < 0.001) than gymnasts and nonathletic controls. When adjusted for body surface area, there was no difference in lean mass between swimmers and gymnasts, and both were higher than controls (p < 0.01). Gymnasts had a lower (p > 0.005) fat mass than swimmers and controls. There were no group differences for spine or whole body BMD, but gymnasts had higher spine BMD corrected for body mass than either swimmers or controls. Gymnasts (1.117 ± 0.110) had higher femoral neck BMD than controls (0.974 ± 0.105), who were higher than swimmers (0.875 ± 0.105) (p = 0.0001). This result still applied when BMD was normalized for body weight and bone size. Trochanter BMD of gymnasts (0.898 ± 0.130) was also higher than controls (0.784 ± 0.097) and swimmers (0.748 ± 0.085) (p = 0.0002), and remained higher when corrected for body mass. Whole body BMD normalized for body mass or bone size was significantly higher in gymnasts than in swimmers. Subregion analysis revealed arm BMD of gymnasts was greater than swimmers and controls (p > 0.01), and gymnasts' leg BMD was higher than that of swimmers (p > 0.05). These results suggest that the intensive weight-bearing activity characteristic of gymnastics is a powerful osteogenic stimulus and that long-term nonweightbearing training that incorporates forceful muscular contractions, as in swimming, confers no beneficial skeletal effects on bone mass of young women.
Mineralization of alkaline phosphatase-complexed collagen implants in the rat in relation to serum inorganic phosphate
Authors: Van Den Bos T. , Oosting J. , Everts V. , Beertsen W.
Published: February 2010

The present study was designed to determine the relationship between mineralization of collagenous matrices and serum levels of calcium and inorganic phosphate. Collagen slices were prepared from bovine dentin or cortical bone and complexed with varying amounts of intestinal alkaline phosphatase (ALP). The enzyme was added to induce de novo mineralization. The ALP-complexed slices were implanted subcutaneously over the skull and in the dorsolateral aspect of the abdominal wall in female Wistar rats of various ages (5-, 10-, 20-, or 35-week-old) and in young male rats fed on a low-P diet. After 1–4 weeks, the implants were removed and analyzed for calcium and phosphate content. In addition, serum levels of calcium and phosphate (total and inorganic) were determined. It was shown that the highest mineral influx occurred in the younger rats (which were also highest in serum Pi), whereas almost no mineral uptake occurred in the older ones. Also in rats fed on a low-P diet (which were low in serum Pi), a strongly decreased mineral influx was noted. In all animal groups a positive correlation was found between the degree of mineralization and serum Pi. No distinct relationship was found between serum Ca/organic phosphate levels and mineral influx in the implants. In vitro incubation of ALP-collagen conjugates in serum from younger and older rats confirmed our view that serum Pi, besides local levels of ALP, is important in de novo mineral deposition. For accretion of mineral in partially remineralized collagenous carriers, ALP activity was not required.
Characterization of an Avian Bone Sialoprotein (BSP) cDNA: Comparisons to Mammalian BSP and Identification of Conserved Structural Domains
Authors: Yang Renji , Gotoh Yozo , Moore Mark A. , Rafidi Krishna , Gerstenfeld Louis C.
Published: February 2010

Bone sialoprotein (BSP) is one of the major noncollagenous proteins found in mineralized vertebrate tissue. It is an acidic glycoprotein containing a high sialic acid content and is phosphorylated on several of its Ser and Thr residues. While it has been extensively characterized from various mammalian species, little is known about its sequence or expression in lower vertebrates. The cloning and characterization of several cDNAs encoding the chicken bone sialoprotein are reported here. A partial cDNA clone encoding the carboxyl terminus of the protein was initially isolated from a λGT11 expression library using a polyclonal antibody against BSP purified from chicken bone matrix. Subsequently, several additional clones were obtained by further screening and by reverse transcription polymerase chain reaction (RT-PCR). Three overlapping clones encompassing about 1 kb, which included the complete coding sequence for BSP, were analyzed. The deduced amino acid sequence revealed that chicken BSP contains 276 amino acid residues. Although the overall identity between chicken and mammalian BSP is only ∼39%, the diversity in amino acid sequence occurs mostly between the major functional domains of this molecule. These domains include: (1) three acidic poly-Glu regions; (2) two tyrosine-rich domains, which may be sites for protein sulfation; (3) several casein kinase II phosphorylation sites; (4) an Asn glycosylation site; and (5) an RGD cell-binding motif. Of interest in the chicken BSP is the identification of two additional RGD motifs within the avian sequence, unlike the mammalian forms of BSP which has only one. The existence of several consecutive gly-x-y triplets in the molecule suggest that it may bind to collagen, which is consistent with the fact that BSP protein is tightly associated with the matrix. Northern blot analysis demonstrated that chicken BSP was expressed specifically in bone tissue, and its expression was detected as early as Day 12 during chicken embryogenesis. BSP expression preceded osteopontin (OPN) expression, reaching maximal levels during embryogenesis in contrast to that of OPN, which only reaches its highest level of expression several weeks after hatching.