bluuebunny/crossref_metadata_2025 huggingface dataset:
Precision and Accuracy of Peripheral Quantitative Computed Tomography (pQCT) in the Mouse Skeleton Compared With Histology and Microcomputed Tomography (μCT)
Authors: Schmidt Corina , Priemel Matthias , Kohler Thomas , Weusten Axel , Müller Ralph , Amling Michael , Eckstein Felix
Published: April 2006

pQCT was evaluated for accuracy of phenotypic characterization of mouse bone in vivo. Bones (tibia, femur, spine) of 27 animals were measured ex vivo with pQCT, μCT, and histomorphometry and of 23 mice in vivo (pQCT). pQCT yielded satisfactory in vivo precision and accuracy in skeletal characterization.

Introduction: Important aspects of modern skeletal research depend on the phenotypic characterization of genetically manipulated mice, with some approaches requiring in vivo measurement. Peripheral quantitative computed tomography (pQCT) is applicable in vivo and provides opportunities to determine a large variety of bone parameters. Here we test the ex vivo and in vivo reproducibility of pQCT, and its accuracy in comparison with histomorphometry and microcomputed tomography (μCT).

Materials and Methods: We examined the tibia, femur, and lumbar spine of 27 mice ex vivo with high-resolution pQCT, using two mouse models (wild-type and ob/ob) with known differences in bone density. Measurements were repeated three times at different days in nine animals. In a second experiment, 23 animals (10 wild-type and 13 bGH transgenic mice) were repeatedly measured in vivo at 12 and 13 weeks of age, respectively.

Results: Among metaphyseal sites, the ex vivo precision was highest at the distal femur (RMS CV <1% for density and <2% for area). The correlation between density (pQCT) and bone volume fraction (histomorphometry) was r2 = 0.79 (tibia, femur, and spine), and that with μCT was r2 = 0.94 (femur). At the diaphysis, the precision was highest at the femur (<2% for total and cortical area), and the correlation with μCT was r2 > 0.77. The in vivo precision for bone density (distal femur) was 2.3–5.1%, and that for absolute and relative cortical area (tibia) was 3.1% and 2.2%.

Conclusions: The results show that pQCT can yield satisfactory precision and accuracy in skeletal characterization of mouse bones, if properly applied. The potential advantage of pQCT is that it provides a large set of parameters on bone properties and that it can be used in vivo, extending the available methodological repertoire for genetic studies.
An Investigation of Unique and Shared Gene Effects on Speed of Sound and Bone Density Using Axial Transmission Quantitative Ultrasound and DXA in Twins
Authors: Knapp Karen M , Andrew Toby , MacGregor Alex J , Blake Glen M , Fogelman Ignac , Spector Tim D
Published: April 2006

The genetic influences of speed of sound (SOS) and BMD were evaluated using 215 pairs of healthy female twins. Genetic influences were found for all SOS and BMD measurements. A combination of shared and unique genetic influences was found to control BMD and SOS at the radius.

Introduction: The aim of this study was to investigate to what extent axial transmission speed of sound (SOS) measurements in cortical bone at multiple, peripheral skeletal sites will be influenced by genetic factors and to estimate the proportion of shared and unique genetic influences controlling bone mineral density (BMD) and SOS at a single site, the radius.

Materials and Methods: The study population consisted of 215 pairs of healthy female twins. Of these, 85 pairs were monozygotic (MZ) and 130 pairs were dizygotic (DZ). The twins had measurements of the nondominant third proximal phalanx, one-third radius, midshaft tibia, and fifth metatarsal using the Sunlight Omnisense and DXA measurements of the lumbar spine, nondominant proximal femur, nondominant radius, and whole body using Hologic QDR-4500W densitometers. Calcaneal quantitative ultrasound (QUS) measurements were performed using the McCue CUBA clinical. Intraclass correlations were calculated, and heritability was estimated using multiple regression analysis. Bivariate analysis of site-matched SOS and BMD measurements at the radius was performed using a variance components analysis program.

Results: Age- and body mass index-adjusted heritability estimates ranged from 0.51 (95% CI, 0.32–0.70) to 0.56 (0.37–0.76) for SOS measurements, 0.58 (0.41–0.75) for broadband ultrasound attenuation (BUA), 0.72 (0.58–0.86) to 0.77 (0.63–0.91) for axial BMD, and 0.53 (0.16–0.90) to 0.63 (0.26–1.00) for radius and whole body BMD. The correlation between SOS and DXA at the radius was r = 0.34 (0.29–0.47). Thirty-eight percent (16–57%) of the genetic variance explained by SOS at the radius was also explained by BMD (one-third radius region of interest), with 62% being unique.

Conclusion: In conclusion, genetic influences were demonstrated for SOS measurements in cortical bone at multiple sites, axial BMD, calcaneal BUA, radius, and whole body BMD. At the radius, up to 38% of the genetic influence is shared by genes controlling BMD and SOS. Clarifying the site specificity and pleiotropic effects of bone genes should help our understanding of these complex pathways.
Carbonated Soft Drink Consumption and Bone Mineral Density in Adolescence: The Northern Ireland Young Hearts Project
Authors: McGartland C , Robson PJ , Murray L , Cran G , Savage MJ , Watkins D , Rooney M , Boreham C
Published: April 2006

In an observational study of 1335 boys and girls aged 12 and 15 years, higher intakes of carbonated soft drinks (CSDs) were significantly associated with lower bone mineral density at the heel, but only in girls. Owing to the upward trend in CSD intake in adolescence, this finding may be of concern.

Introduction: High consumption of carbonated soft drinks (CSD) during adolescence may reduce bone mineral accrual and increase fracture risk. The aim of this study was to examine the relationship between CSD consumption and bone mineral density (BMD) in a representative sample of adolescents.

Materials and Methods: This was a cross-sectional observational study in 36 postprimary schools in Northern Ireland. Participants included 591 boys and 744 girls either 12 or 15 years old. BMD was measured by DXA, and usual beverage consumption was assessed by the diet history method. Adjusted regression modeling was used to investigate the influence of CSD on BMD.

Results: A significant inverse relationship between total CSD intake and BMD was observed in girls at the dominant heel (β, −0.099; 95% CI, −0.173 to −0.025). Non-cola consumption was inversely associated with dominant heel BMD in girls (β, −0.121; 95% CI, −0.194 to −0.048), and diet drinks were also inversely associated with heel BMD in girls (β, −0.087; 95% CI, −0.158 to −0.016). However, no consistent relationships were observed between CSD intake and BMD in boys. Cola consumption and nondiet drinks were not significantly related to BMD in either sex.

Conclusion: CSD consumption seems to be inversely related to BMD at the dominant heel in girls. It is possible that the apparent association results from the displacement of more nutritious beverages from the diet. Although the inverse association observed between CSD consumption and BMD is modest and confined to girls, this finding may have important public health implications given the widespread use and current upward trend in CSD consumption in Western populations.
Characterization of Bone Structure in Leptin Receptor-Deficient Zucker (fa/fa) Rats
Authors: Tamasi Joseph A , Arey Brian J , Bertolini Donald R , Feyen Jean HM
Published: April 2006

To investigate the role of leptin in bone formation, the skeleton of the obese female leptin receptor-deficient Zucker rat was examined using pQCT, μCT, and histomorphometry. A trend toward decreasing structural and bone formation parameters in these rats as they age suggest that leptin has a small positive effect on bone.

Introduction: Evidence in the literature has suggested the possible role of leptin in bone formation. Leptin deficiency or leptin receptor deficiency results in higher bone mass. In an attempt to further investigate leptin's role in bone formation, we examined the skeleton of obese leptin receptor-deficient Zucker rats.

Methods: Female leptin receptor-deficient Zucker (fa/fa) rats and their homozygous (Fa/Fa) and heterozygous (Fa/fa) lean controls were used at 9 and 15 weeks of age (n = 5). Bone mineral density of the proximal tibia was measured by peripheral quantitative computed tomography (pQCT). Microcomputed tomography (μCT) was used for the analysis of trabecular architecture in the proximal tibia metaphysis and cortical bone at the tibia-fibula junction. Static and dynamic parameters of bone resorption and formation were quantitated by histomorphometry. Statistical analysis was performed by Dunnett's one-way ANOVA.

Results: Analysis of the proximal tibia by pQCT show no significant differences in the bone mineral density of obese rats compared with their corresponding lean controls in either age group. Trabecular architecture measured by μCT indicate a trends toward decreasing bone volume (BV/TV) in the obese animals, evident by a decrease in trabecular number and thickness with an increase in trabecular separation. Histomorphometric evaluation further shows significant increases in osteoclast surface in the obese rats at both 9 and 15 weeks without a change in osteoclast number. Osteoid surface in the obese animals was also found to be decreased by 15 weeks of age. Fluorescent-based measurements of bone formation were not significantly different. Differences in the cortical compartment were not observed at either age.

Conclusion: Based on the observed skeletal phenotype of the Zucker (fa/fa) rat, it is suggested that leptin exerts a positive effect on bone.
Determinants of Bone Density in 30- to 65-Year-Old Women: A Co-Twin Study
Authors: MacInnis RJ , Cassar C , Nowson CA , Paton LM , Flicker L , Hopper JL , Larkins RG , Wark JD
Published: April 2006

Reported effects of body composition and lifestyle on bone mineral density in pre-elderly adult women have been inconsistent. In a co-twin study, we measured bone mineral density, lean and fat mass, and lifestyle factors. Analyzing within pair differences, we found negative associations between bone mineral density and tobacco use (2.3–3.3% per 10 pack-years) and positive associations with sporting activity and lean and fat mass.

Introduction: Reported effects of body composition and lifestyle of bone mineral density in pre-elderly adult women have been inconsistent.

Methods: In a co-twin study of 146 female twin pairs aged 30 to 65 years, DXA was used to measure bone mineral density at the lumbar spine, total hip, and forearm, total body bone mineral content, and lean and fat mass. Height and weight were measured. Menopausal status, dietary calcium intake, physical activity, current tobacco use, and alcohol consumption were determined by questionnaire. Within-pair differences in bone measures were regressed through the origin against within-pair differences in putative determinants.

Results: Lean mass and fat mass were associated with greater bone mass at all sites. A discordance of 10 pack-years smoking was related to a 2.3–3.3% (SE, 0.8–1.0) decrease in bone density at all sites except the forearm, with the effects more evident in postmenopausal women. In all women, a 0.8% (SE, 0.3) difference in hip bone mineral density was associated with each hour per week difference in sporting activity, with effects more evident in premenopausal women. Daily dietary calcium intake was related to total body bone mineral content and forearm bone mineral density (1.4 ± 0.7% increase for every 1000 mg). Lifetime alcohol consumption and walking were not consistently related to bone mass.

Conclusion: Several lifestyle and dietary factors, in particular tobacco use, were related to bone mineral density. Effect sizes varied by site. Characterization of determinants of bone mineral density in midlife and thereafter may lead to interventions that could minimize postmenopausal bone loss and reduce osteoporotic fracture risk.
Height and Lower Extremity Length as Predictors of Hip Fracture: Results of the NHANES I Epidemiologic Follow-up Study
Authors: Opotowsky Alexander R , Su Brian W , Bilezikian John P
Published: April 2006

The relationship between height, lower extremity length (LEL), and hip fracture was examined in 4264 women followed for 22 years. Greater height predicted fracture among younger women, whereas LEL predicted risk for all ages. LEL is a better predictor of hip fracture risk than height among older women.

Introduction: The significance of height as a risk factor for hip fracture is controversial. Age-related height loss, caused by nonpathological processes and vertebral deformities, may confound the relationship between standing height and hip fracture. In contrast, LEL might be less subject to age-related changes and thus be a better index of fracture risk for older individuals.

Methods: We evaluated data available from 4264 white women who participated in the NHANES I Epidemiologic Follow-up Survey. There were 203 incident hip fractures over the 22-year follow-up period. The effect of height and LEL on hip fracture risk was determined for three age groups: 40–59, 60–69, and 70–74 year olds.

Results: After controlling for potential confounders, the hazard ratio (HR) for hip fracture for each SD greater standing height was 1.81 for the youngest age group (p = 0.002), 1.29 for 60–69 year olds (p = 0.02), and 1.10 for 70–74 year olds (p = 0.49). In contrast, LEL predicted hip fracture risk for all age groups, with HRs of 1.43 (p = 0.02), 1.30 (p = 0.002), and 1.33 (p = 0.02), respectively. Similarly, higher tertile of standing height did not confer increased hip fracture risk among the oldest women, but higher tertile of LEL did. Among women that were 70–74 years old, those in the highest tertile of LEL had a 1.83-fold (p < 0.05) greater risk of hip fracture than those in the lowest tertile.

Conclusions: The results support the hypothesis that LEL is a better predictor of hip fracture than standing height among older women, the group at highest risk for these fractures.
Abnormalities in Bone Mineral Density and Bone Histology in Thalassemia
Authors: Domrongkitchaiporn Somnuek , Sirikulchayanonta Vorachai , Angchaisuksiri Pantep , Stitchantrakul Wasana , Kanokkantapong Chavasak , Rajatanavin Rajata
Published: April 2006

This study demonstrated that there was extensive iron staining on trabecular surface and marked reduction in trabecular bone volume without significant alteration in bone formation and bone resorption rates as well as significant reduction in bone mineral density in 18 thalassemic patients. Serum IGF-I was reduced and may modulate the reduction of bone mass.

Introduction: Bone histomorphometric studies in thalassemia to show alterations in bone histology and their relationship to biochemical parameters are very limited. Therefore, this study was systematically conducted to determine the alterations in thalassemia patients.

Methods: Serum biochemical parameters, trans-iliac crest bone biopsy, and determination of bone mineral density of femur and lumbar spine were done in 18 thalassemic patients (10 females and 8 males).

Results: Serum osteocalcin, carboxy terminal teleopeptide fragment of type I collagen, and parathyroid hormone levels were within normal limits, but serum 25(OH) vitamin D (19.3 ± 1.6 ng/ml) and 1,25(OH)2 vitamin D (33.77 ± 1.51 pg/ml) levels were decreased. Serum insulin-like growth factor I (IGF-I; 145.2 ± 20 ng/ml) was suppressed, whereas serum ferritin (1366.6 ± 253.9 ng/ml) was markedly elevated. Reduced bone mineral density was found in all studied areas. Trabecular bone volume was significantly decreased (16.65 ± 1.12%), whereas bone formation rate, eroded surface, and other bone histomorphometric parameters were within normal limits. The trabecular bone volume varied significantly with bone mineral density of total femur (r = 0.48, p = 0.04). There was an extensive stainable iron surface on the mineral front (9–60%). Significant correlation between serum IGF-I, serum ferritin, stainable iron surface, and bone mineral density, lumbar spine, and total femur were found. Serum IGF-I correlated with trabecular bone volume (r = 0.6, p = 0.03), inversely with both serum ferritin level (r = −0.6, p < 0.01), and inversely with stainable iron surface (r = −0.53, p = 0.02). Multiple regression analysis demonstrated that IGF-I was the only independent variable that determined bone mineral density of lumbar spine and total femur.

Conclusion: Low bone mineral density and reduced trabecular bone volume with extensive iron deposition are the predominant findings in thalassemic patients. There was no evidence of increased bone resorption or mineralization defect. A reduction in circulatory IGF-I may modulate the reduction of bone mass.
Osteopontin Expression in Osteoblasts and Osteocytes During Bone Formation Under Mechanical Stress in the Calvarial Suture In Vivo
Authors: Morinobu Mikihiko , Ishijima Muneaki , Rittling Susan R , Tsuji Kunikazu , Yamamoto Haruyasu , Nifuji Akira , Denhardt David T , Noda Masaki
Published: April 2006

To clarify the role of OPN in bone formation under mechanical stress, we examined the expression and the function of OPN in bone using an expansion force-induced osteogenesis model. Our results indicated that OPN expression was enhanced during the bone formation and that OPN would be one of the positive factors for the bone formation under mechanical stress.

Introduction: Bone formation is known to be stimulated by mechanical stress; however, molecules involved in stress-dependent regulation of bone formation have not yet been fully characterized. Extracellular matrix proteins such as osteopontin (OPN) could play a role in mediation of the mechanical stress signal to osteoblasts. However, the function of OPN in bone formation under mechanical force is not known. Therefore, we examined the expression and the role of OPN in bone formation in vivo under tensile mechanical stress.

Materials and Methods: Sagittal sutures of mice were subjected to expansion mechanical stress by setting orthodontic spring wires, and OPN expression during bone formation within the suture gap was examined.

Results: Expansion of the sutures resulted in bone formation at the edges of the parietal bones within the sagittal suture. Immunohistochemical analysis revealed abundant accumulation of OPN protein in the matrix of newly formed bone on the inner edge of the parietal bone within the mechanically expanded sutures. Osteoblasts forming bone within the suture subjected to tensile stress also exhibited high levels of OPN protein expression. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that OPN mRNA expression was enhanced in wild-type calvariae subjected to expansion force compared with the control calvariae where dead spring wires were set without expansion stress. In addition, type I collagen mRNA was also expressed in the calvariae under the mechanical stimuli. To understand the function of OPN, sagittal sutures in OPN-deficient mice were subjected the expansion stress, and bone formation within the suture to fill the expanded gap was compared with that observed in wild-type mice. OPN deficiency reduced bone formation at the edge of the parietal bone in contact with the expanded suture gap.

Conclusions: These observations revealed that OPN plays a pivotal role in bone formation under tensile mechanical stress.
Identification of Biphenylcarboxylic Acid Derivatives as a Novel Class of Bone Resorption Inhibitors
Authors: van 't Hof Rob J , Idris Aymen I , Ridge Susan A , Dunford James , Greig Iain R , Ralston Stuart H
Published: April 2006

A novel class of biphenylcarboxylic acid derivatives are described that inhibit osteoclastic bone resorption in vitro by promoting osteoclast apoptosis and that prevent ovariectomy-induced bone loss in vivo. The compounds act by a novel mechanism that seems to be distinct from existing antiresorptive drugs.

Introduction: Many common bone diseases such as osteoporosis, Paget's disease, and cancer-associated bone disease are characterized by excessive bone loss caused by increased osteoclastic activity. Successful treatment of these diseases is based on osteoclast inhibition. The osteoclast inhibitory drugs that are currently available fall into relatively few mechanistic classes, indicating the need to identify novel antiresorptives. Here we describe a series of biphenylcarboxylic acid derivatives that have potent inhibitory effects on osteoclastic bone resorption in vitro and on ovariectomy-induced bone loss in vivo.

Materials and Methods: Compounds were tested for inhibitory effects on bone resorption in vitro using mouse osteoblast-bone marrow co-cultures, isolated rabbit osteoclasts, and mouse osteoclasts generated from bone marrow. Some experiments were also performed on human osteoclasts generated from peripheral blood mononuclear cells. We also investigated the effects of specific compounds on ovariectomy-induced bone loss in vivo in mice.

Results: One of the most potent compounds identified was the butanediol ester of biphenyl carboxylic acid (ABD056), which inhibited osteoclast formation in mouse osteoblast-bone marrow co-cultures by 50% (IC50) at a concentration of 26 μM and in macrophage-colony stimulating factor (M-CSF)- and RANKL-stimulated mouse bone marrow cultures with an IC50 of 8 μM. Mechanistic studies showed that ABD056 caused osteoclast apoptosis and inhibited TNFα-induced NF-κB activation. No inhibitory effects on osteoblast growth or differentiation were observed at concentrations of up to 100 μM. When administered to mice at doses of 5 and 10 mg/kg/day, ABD056 prevented ovariectomy-induced bone loss.

Conclusions: Butanediol biphenylcarboxylic acid derivatives represent a new class of antiresorptive drug that might be of therapeutic value in the prevention and treatment of diseases characterized by osteoclast activation such as osteoporosis, cancer-associated bone disease, and Paget's disease of bone.
Localization of the Gene Causing the Osteopetrotic Phenotype in the Incisors Absent (Ia) Rat on Chromosome 10q32.1
Authors: van Wesenbeeck Liesbeth , Odgren Paul R , Mackay Carole A , Van Hul Wim
Published: April 2006

The incisors absent rat is an osteopetrotic animal model. Segregation analysis in 37 affected animals from an outcross enabled us to assign the disease causing gene to a 4.7-cM interval on rat chromosome 10q32.1. Further analysis of the genes mapped in this region will provide more insight into the underlying pathogenesis.

Introduction: Many of the insights into the factors that regulate the differentiation and activation of osteoclasts are gained from different spontaneous and genetically induced osteopetrotic animal models. The osteopetrotic incisors absent (ia) rat exhibits a generalized skeletal sclerosis and a delay of tooth eruption. Although the ia rat has well been studied phenotypically, the genetic defect still remains unknown.

Material and Methods: To map the ia locus, we outcrossed the inbred ia strain with the inbred strain Brown Norway. Intercrossing F1 animals produced the F2 generation. Thirty-one mutant F2 animals and six mutant F4 animals were available for segregation analysis.

Results: Segregation analysis enabled us to assign the disease causing gene to rat chromosome 10q32.1. Homozygosity for the ia allele was obtained for two of the markers analyzed (D10Rat18 and D10Rat84). Key recombinations delineate a candidate region of 4.7 cM flanked by the markers D10Rat99 and D10Rat17.

Conclusion: We have delineated a 4.7-cM region on rat chromosome 10q32.1 in which the gene responsible for the osteopetrotic phenotype of the ia rat is located. Although the sequence of this chromosomal region is not complete, over 140 known or putative genes have already been assigned to this region. Among these, several candidate genes with a putative role in osteoclast functioning can be identified. However, at this point, it cannot be excluded that one of the genes with a currently unknown function is involved in the pathogenesis of the ia rat. Further analysis of the genes mapped in this region will provide us more insight into the pathogenesis of this osteopetrotic animal model.
Negative Regulation of Chondrocyte Differentiation by Transcription Factor AP-2α
Authors: Huang Zhengmin , Xu Haiming , Sandell Linda
Published: April 2006

This study investigated the role of transcription factor AP-2α in chondrocyte differentiation in vitro. AP-2α mRNA declined during differentiation, and overexpression of AP-2α inhibited differentiation. The results demonstrated that AP-2α plays a negative role in chondrocyte differentiation.

Introduction: Transcription factor AP-2α has been detected in growth plate and articular chondrocytes and has been shown to regulate cartilage matrix gene expression in vitro. However, the precise functional role of AP-2α in chondrocyte differentiation is not known. In this study, we assessed the expression and the function of AP-2α in chondrocyte differentiation of ATDC5 cells.

Materials and Methods: Chondrocyte differentiation of ATDC5 cells was induced with insulin or transforming growth factor β (TGF-β). Proteoglycan production was assessed by alcian blue staining, and expression levels of chondrocyte marker genes and AP-2 gene family were determined by quantitative real time reverse transcriptase-polymerase chain reaction (RT-PCR). Overexpression of AP-2α in ATDC5 cells was accomplished by retroviral infection. Infected cells were selected for G418 resistance and pooled for further analysis.

Results and Conclusions: Quantitative real time RT-PCR analysis showed that among the four members of the AP-2 gene family, AP-2α mRNA was the most abundant. AP-2α mRNA levels progressively declined during the differentiation induced by either insulin or TGF-β treatment. Retroviral expression of AP-2α in ATDC5 cells prevented the formation of cartilage nodules, suppressed the proteoglycan production, and inhibited the expression of type II collagen, aggrecan, and type X collagen. Expression profile analysis of key transcription factors involved in chondrogenesis showed that overexpression of AP-2α maintained the expression of Sox9 but suppressed the expression of Sox5 and Sox6. Taken together, we provide, for the first time, molecular and cellular evidence suggesting that AP-2α is a negative regulator of chondrocyte differentiation.
Parathyroid Cells Cultured in Collagen Matrix Retain Calcium Responsiveness: Importance of Three-Dimensional Tissue Architecture
Authors: Ritter Cynthia S , Slatopolsky Eduardo , Santoro Samuel , Brown Alex J
Published: April 2006

Primary cultures of bovine parathyroid cells rapidly lose calcium responsiveness. Here, we show that bovine parathyroid cells grown in collagen coalesce into an organoid (“pseudogland”) with stable calcium responsiveness. These findings also illustrate the importance of 3-D cellular architecture in parathyroid gland function.

Introduction: The ability of extracellular calcium to suppress parathyroid hormone (PTH) secretion is quickly lost in primary monolayer cultures of bovine parathyroid cells. This has been attributed to a decrease in the expression of the cell surface calcium-sensing receptor (CaR), but other factors, including normal cell-to-cell interaction, may be critical. Here we describe a novel system for culturing bovine parathyroid cells that promotes re-formation of a three-dimensional (3-D) cellular architecture and re-establishment of calcium responsiveness.

Materials and Methods: Dispersed bovine parathyroid cells were cultured as monolayers or were mixed with type I collagen and placed in culture plates. CaR mRNA and the calcium regulation of PTH secretion were measured over a period of several weeks in parathyroid cells cultured both in collagen matrix and as monolayers. Calcium regulation of PTH mRNA was also investigated.

Results and Conclusions: Within 1–2 weeks in collagen culture, parathyroid cells coalesced into a small mass approximately 1–2 mm in size (referred to as a pseudogland). Suppression of PTH secretion by high calcium was blunted at 1 day in collagen, but returned within 1 week, and was retained through 3 weeks; the calcium set point (1.05 ± 0.04 mM) was similar to that reported for freshly dispersed cells. PTH mRNA was also suppressed by increasing extracellular calcium. CaR mRNA expression was decreased at 1 day in collagen and increased with time in culture, although never reaching the level found in dispersed cells. In bovine parathyroid cells cultured as monolayers, however, suppression of PTH by calcium was observed only at day 1 in culture. CaR mRNA content fell by 70% at day 1 but remained stable thereafter. Thus, a total loss of calcium responsiveness in monolayers was observed despite significant residual expression of CaR, suggesting that loss of the calcium response cannot be attributed solely to decreased CaR. In summary, the pseudogland model illustrates the importance of the 3-D cellular architecture in parathyroid gland function and provides a useful model in which to investigate calcium-mediated control of parathyroid gland functions, especially those requiring extended treatment.
A Unique Insertion/Substitution in Helix H1 of the Vitamin D Receptor Ligand Binding Domain in a Patient With Hereditary 1,25-Dihydroxyvitamin D-Resistant Rickets
Authors: Malloy Peter J , Xu Rong , Cattani Andreina , Reyes M Loreto , Feldman David
Published: April 2006

A young Chilean boy with severe rickets was found to have hereditary vitamin D-resistant rickets without alopecia. He had a unique insertion/substitution mutation in the ligand-binding domain of the vitamin D receptor. The in-frame mutation disrupted ligand binding and co-activator binding and resulted in 1,25(OH)2D3 resistance.

Introduction: Hereditary vitamin D-resistant rickets (HVDRR) is a genetic disorder caused by mutations in the vitamin D receptor (VDR). In this study, we examined the VDR in a young boy who exhibited the typical clinical features of HVDRR but without alopecia.

Materials and Methods: The patient's VDR was studied using cultured dermal fibroblasts, and the recreated mutant VDR was analyzed in transfected cells.

Results: The patient's fibroblasts were resistant to 1,25-dihydroxyvitamin D [1,25(OH)2D3], exhibiting only a slight induction of 24-hydroxylase gene expression when treated with 1 μM 1,25(OH)2D3. [3H]1,25(OH)2D3 binding was absent in cell extracts from the patient's fibroblasts. Sequence analysis of the VDR gene uncovered a unique 5-bp deletion/8-bp insertion in exon 4. The mutation in helix H1 of the ligand-binding domain deletes two amino acids (H141 and T142) and inserts three amino acids (L141, W142, and A143). In transactivation assays, the recreated mutant VDR was 1000-fold less active than the wildtype (WT) VDR. In glutathione S-transferase (GST) pull-down assays, the mutant VDR bound GST-retinoid X receptor (RXR) weakly in the absence of 1,25(OH)2D3; however, the binding did not increase with increasing concentrations of ligand. The mutant VDR did not bind to GST-vitamin D receptor interacting protein (DRIP) 205 at concentrations up to 1 μM 1,25(OH)2D3. We also examined effects of the three individual mutations on VDR transactivation. Only the insertion of A143 into the WT VDR disrupted VDR transactivation to the same extent observed with the natural mutation.

Conclusion: We describe a novel insertion/substitution mutation in helix H1 of the VDR ligand-binding domain (LBD) that abolishes ligand binding and result in the syndrome of HVDRR. This is the first time an insertion/substitution has been found as the defect-causing HVDRR.
Osteoblast-Like Cells From Estrogen Receptor α Knockout Mice Have Deficient Responses to Mechanical Strain
Authors: Jessop Helen L , Suswillo Rosemary FL , Rawlinson Simon CF , Zaman Gul , Lee Karla , Das-Gupta Vicky , Pitsillides Andrew A , Lanyon Lance E
Published: April 2006

Osteoblast-like cells in primary cultures derived from ERα−/− mice do not proliferate in response to mechanical strain, unlike those from their ERα+/+ littermates. ERα−/− cells also lack strain-related NO production and responsiveness to IGFs. Proliferative responsiveness to strain is rescued by transfection with functional ERα. ERα number or function in bone cells may limit bones' adaptability to mechanical loading.

Introduction: In vivo, bones' osteogenic response to mechanical loading involves proliferation of surface osteoblasts. This response is replicated in vitro and involves ERK-mediated activation of the estrogen receptor (ER) α and upregulation of estrogen response element activity. This proliferative response can be blocked by selective estrogen receptor modulators and increased by transfection of additional ERα.

Materials and Methods: We have now investigated the mechanisms of ER involvement in osteoblast-like cells' early responses to strain by comparing the responses of primary cultures of these cells derived from homozygous ERα knockout (ERKO) mice (ERα−/−) with those from their wildtype (ERα+/+) and heterozygous (ERα+/−) littermates and from ERβ knockout (BERKO) mice (ERβ+/+, ERβ+/−, and ERβ−/−).

Results: Whereas ERα+/+, ERα+/−, ERβ+/+, and ERβ−/− cells proliferate in response to a single 10-minute period of cyclic strain, ERα−/− cells do not. Transfection of fully functional, but not mutant, ERα rescues the proliferative response to strain in these cells. The strain-related response of ERα−/− cells is also deficient in that they show no increased activity of an AP-1 driven reporter vector and no strain-related increases in NO production. Their strain-related increase in prostacyclin production is retained. They proliferate in response to fibroblast growth factor-2 but not insulin-like growth factor (IGF)-I or IGF-II, showing the importance of ERα in the IGF axis and the ability of ERα−/− cells to proliferate normally in response to a mitogenic stimulus that does not require functional ERα.

Conclusions: These data indicate ERα's obligatory involvement in a number of early responses to mechanical strain in osteoblast-like cells, including those that result in proliferation. They support the hypothesis that reduction in ERα expression or activity after estrogen withdrawal results in a less osteogenic response to loading. This could be important in the etiology of postmenopausal osteoporosis.
Bone Morphogenetic Protein 2 Induces Cyclo-oxygenase 2 in Osteoblasts via a Cbfa1 Binding Site: Role in Effects of Bone Morphogenetic Protein 2 In Vitro and In Vivo
Authors: Chikazu Daichi , Li Xiaodong , Kawaguchi Hiroshi , Sakuma Yoko , Voznesensky Olga S. , Adams Douglas J. , Xu Manshan , Hoshi Kazuto , Katavic Vedran , Herschman Harvey R. , Raisz Lawrence G. , Pilbeam Carol C.
Published: April 2006

We tested the hypothesis that induction of cyclo-oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP-2 induced COX-2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP-2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX-2 promoter-luciferase reporter construct (Pluc) and in MC3T3-E1 cells transfected with Pluc. Deletion analysis identified the -300/-213-bp region of the COX-2 promoter as necessary for BMP-2 stimulation of luciferase activity. Mutation of core-binding factor activity 1 (muCbfa1) consensus sequence (5′-AACCACA-3′) at -267/-261 bp decreased BMP-2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfa1 site was inhibited or supershifted by specific antibodies to Cbfa1. In cultured osteoblasts from calvariae of COX-2 knockout (-/-) and wild-type (+/+) mice, the absence of COX-2 expression reduced the BMP-2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long bones, BMP-2 induced osteoclast formation in cells from COX-2+/+ mice but not in cells from COX-2−/− mice. In vivo, BMP-2 (10 μg/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (μCT), was decreased by 78% in COX-2−/− mice compared with COX-2+/+ mice. We conclude that BMP-2 transcriptionally induces COX-2 in osteoblasts via a Cbfa1 binding site and that the BMP-2 induction of COX-2 can contribute to effects of BMP-2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP-2 stimulation of ectopic bone formation in vivo.
Suppressed Bone Turnover by Long-Term Bisphosphonate Treatment Accumulates Microdamage but Maintains Intrinsic Material Properties in Cortical Bone of Dog Rib
Authors: Komatsubara Satoshi , Mori Satoshi , Mashiba Tasuku , Li Jilliang , Nonaka Kiichi , Kaji Yoshio , Akiyama Tomoyuki , Miyamoto Kensaku , Cao Yongping , Kawanishi Jun , Norimatsu Hiromichi
Published: April 2006

Effects of long-term suppression of bone remodeling by bisphosphonate were investigated in cortical bone of dog rib. Although microdamage was accumulated, BMD was increased without increasing cortical bone area. Consequently, the intrinsic material properties were not reduced.

Introduction: Recently, we have reported that long-term suppression of bone remodeling increases microdamage accumulation but is not necessarily associated with vertebral fragility because of compensated increase of bone mass and improved microarchitecture. This study aimed to investigate the effect of long-term suppression of bone remodeling by bisphosphonate on the degree of mineralization, accumulation of microdamage, and mechanical properties of cortical bone in the same dogs.

Materials and Methods: Twenty-nine 1-year-old beagles (15 males, 14 females) were divided into three groups and treated daily with vehicle (CNT) or with incadronate at a dose of 0.3 (LOW) or 0.6 mg/kg/day (HIGH) orally for 3 years. After death, pQCT, histomorphometry, microdamage measurements, and three-point bending mechanical test were performed using the ninth rib.

Results: Cortical BMD was increased in the incadronate-treated groups. Cortical activation frequency was suppressed by 82% and 70% in HIGH and LOW, respectively, compared with CNT, without impairment of mineralization. Microdamage accumulation was increased in both incadronate-treated groups. Although there were no significant differences in total and cortical area among the three groups, structural mechanical properties were significantly increased after incadronate treatment while intrinsic material properties were not changed in the incadronate-treated groups.

Conclusion: This study suggests that long-term suppression of bone remodeling by bisphosphonate increases microdamage accumulation. However, this was not necessarily associated with a reduction of intrinsic material properties probably because of an increased degree of mineralization.
A Single-Dose Placebo-Controlled Study of AMG 162, a Fully Human Monoclonal Antibody to RANKL, in Postmenopausal Women
Authors: Bekker Pirow J , Holloway Donna L , Rasmussen Amy S , Murphy Robyn , Martin Steven W , Leese Philip T , Holmes Gregory B , Dunstan Colin R , DePaoli Alex M
Published: April 2006

The safety and bone antiresorptive effect of a single subcutaneous dose of AMG 162, a human monoclonal antibody to RANKL, was investigated in 49 postmenopausal women. AMG 162 is a potent antiresorptive agent for diseases such as osteoporosis.

Introduction: RANKL is an essential osteoclastic differentiation and activation factor.

Materials and Methods: The bone antiresorptive activity and safety of AMG 162, a fully human monoclonal antibody to RANKL, were evaluated in postmenopausal women in this randomized, double-blind, placebo-controlled, single-dose, dose escalation study. Six cohorts of eight to nine women were randomly assigned to receive a single subcutaneous injection of either AMG 162 or placebo (3:1 ratio). AMG 162 doses were 0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 mg/kg. Subjects were followed up to 6 months in all cohorts and 9 months in the three highest dose cohorts. Second morning void urinary N-telopeptide/creatinine (NTX; Osteomark), serum NTX, and serum bone-specific alkaline phosphatase (BALP, Ostase) were assessed as bone turnover markers.

Results and Conclusions: Forty-nine women were enrolled. A single subcutaneous dose of AMG 162 resulted in a dose-dependent, rapid (within 12 h), profound (up to 84%), and sustained (up to 6 months) decrease in urinary NTX. At 6 months, there was a mean change from baseline of −81% in the 3.0 mg/kg AMG 162 group compared with −10% in the placebo group; serum NTX changes were −56% and 2%, respectively. BALP levels did not decrease remarkably until after 1 month, indicating that the effect of AMG 162 is primarily antiresorptive. Intact parathyroid hormone (PTH) levels increased up to ∼3-fold after 4 days in the 3.0 mg/kg dose group, but returned toward baseline with follow-up. Albumin-adjusted serum calcium did not decrease >10% on average in any group, and no subject had values beiow 2 mmol/liter. AMG 162 was well tolerated. No related serious adverse events occurred. No clinically meaningful laboratory changes, other than those described above, were observed. In summary, a single subcutaneous dose of AMG 162 resulted in a dose-dependent rapid and sustained decrease from baseline in bone turnover and could be an effective and convenient treatment for osteoporosis.
Nitrogen-Containing Biphosphonates Inhibit the Mevalonate Pathway and Prevent Post-Translational Prenylation of GTP-Binding Proteins, Including Ras
Authors: Luckman Steven P. , Hughes David E. , Coxon Fraser P. , Russell R. Graham G. , Dr. Rogers Michael J.
Published: April 2006

Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of metabolic bone diseases. Although the molecular targets of bisphosphonates have not been identified, these compounds inhibit bone resorption by mechanisms that can lead to osteoclast apoptosis. Bisphosphonates also induce apoptosis in mouse J774 macrophages in vitro, probably by the same mechanisms that lead to osteoclast apoptosis. We have found that, in J774 macrophages, nitrogen-containing bisphosphonates (such as alendronate, ibandronate, and risedronate) inhibit post-translational modification (prenylation) of proteins, including the GTP-binding protein Ras, with farnesyl or geranylgeranyl isoprenoid groups. Clodronate did not inhibit protein prenylation. Mevastatin, an inhibitor of 3-hydroxy-3-methylglutatyl (HMG)-CoA reductase and hence the bio-synthetic pathway required for the production of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, also caused apoptosis in J774 macrophages and murine osteoclasts in vitro. Furthermore, alendronate-induced apoptosis, like mevastatin-induced apoptosis, could be suppressed in J774 cells by the addition of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, while the effect of alendronate on osteoclast number and bone resorption in murine calvariae in vitro could be overcome by the addition of mevalonic acid. These observations suggest that nitrogen-containing bisphosphonate drugs cause apoptosis following inhibition of post-translational prenylation of proteins such as Ras. It is likely that these potent antiresorptive bisphosphonates also inhibit bone resorption by preventing protein prenylation in osteoclasts and that enzymes of the mevalonate pathway or prenyl protein transferases are the molecular targets of the nitrogen-containing bisphosphonates. Furthermore, the data support the view that clodronate acts by a different mechanism.
Use of Oral Corticosteroids and Risk of Fractures
Authors: Van Staa T. P. , Leufkens H. G. M. , Abenhaim L. , Zhang B. , Cooper C.
Published: April 2006

Treatment with oral corticosteroids is known to decrease bone density but there are few data on the attendant risk of fracture and on the reversibility of this risk after cessation of therapy. A retrospective cohort study was conducted in a general medical practice setting in the United Kingdom (using data from the General Practice Research Database [GPRD]). For each oral corticosteroid user aged 18 years or older, a control patient was selected randomly, who was matched by age, sex, and medical practice. The study comprised 244,235 oral corticosteroid users and 244,235 controls. The average age was 57.1 years in the oral corticosteroid cohort and 56.9 years in the control cohort. In both cohorts 58.6% were female. The most frequent indication for treatment was respiratory disease (40%). The relative rate of nonvertebral fracture during oral corticosteroid treatment was 1.33 (95% confidence interval [CI], 1.29–1.38), that of hip fracture 1.61 (1.47–1.76), that of forearm fracture 1.09 (1.01–1.17), and that of vertebral fracture 2.60 (2.31–2.92). A dose dependence of fracture risk was observed. With a standardized daily dose of less than 2.5 mg prednisolone, hip fracture risk was 0.99 (0.82–1.20) relative to control, rising to 1.77 (1.55–2.02) at daily doses of 2.5–7.5 mg, and 2.27 (1.94–2.66) at doses of 7.5 mg or greater. For vertebral fracture, the relative rates were 1.55 (1.20–2.01), 2.59 (2.16–3.10), and 5.18 (4.25–6.31), respectively. All fracture risks declined toward baseline rapidly after cessation of oral corticosteroid treatment. These results quantify the increased fracture risk during oral corticosteroid therapy, with greater effects on the hip and spine than forearm. They also suggest a rapid offset of this increased fracture risk on cessation of therapy, which has implications for the use of preventative agents against bone loss in patients at highest risk. (J Bone Miner Res 2000;15:993–1000)