bluuebunny/crossref_metadata_2025_split huggingface dataset:
Variation in Bone Biomechanical Properties, Microstructure, and Density in BXH Recombinant Inbred Mice
Authors: Turner Charles H. , Hsieh Yeou-Fang , Müller Ralph , Bouxsein Mary L. , Rosen Clifford J. , McCrann Margaret E. , Donahue Leah Rae , Beamer Wesley G. 
Published: April 2006

Abstract
To test the hypothesis that factors associated with bone strength (i.e., volumetric bone mineral density [vBMD], geometry, and microstructure) have heritable components, we exploited the 12 BXH recombinant inbred (RI) strains of mice derived from C57BL/6J (B6; low bone mass) and C3H/HeJ (C3H; high bone mass) progenitor strains. The femurs and lumbar vertebrae from each BXH RI strain were characterized for phenotypes of vBMD, microstructural, biomechanical, and geometrical properties. Methods included bending (femur) and compression (vertebra) testing, peripheral quantitative computed tomography (pQCT), and microcomputed tomography (μCT). Segregation patterns of femoral and vertebral biomechanical properties among the BXH RI strains suggested polygenic regulation. Femoral biomechanical properties were strongly associated with femoral width in the anteroposterior (AP) direction and cortical thickness-geometric properties with complex genetic regulation. Vertebral vBMD and biomechanical properties measured in BXH RI strains showed a greater variability than either B6 or C3H progenitors, suggesting both progenitor strains have independent subsets of genes that yield similar vBMD and strength. The μCT and pQCT data suggested that the distribution of vertebral mineral into cortical and trabecular compartments is regulated genetically. Although the B6 and C3H progenitors had similar vertebral strength, their vertebral structures were markedly different: B6 had good trabecular bone structure and modest cortical bone mineral content (BMC), whereas C3H had high cortical BMC combined with a deficiency in trabecular structure. These structural traits segregated independently in the BXH RI strains. Finally, vertebral strength was not correlated consistently with femoral strength among the BXH RI strains, suggesting genetic regulation of bone strength is site specific.
Expression of Functional Gap Junctions and Regulation by Fluid Flow in Osteocyte-Like MLO-Y4 Cells
Authors: Cheng Benxu , Zhao Shujie , Luo Jian , Sprague Eugene , Bonewald Lynda F. , Jiang Jean X. 
Published: April 2006

Abstract
Osteocytes are thought to be mechanosensory cells that respond to mechanical stress by sending signals to other bone cells to initiate bone remodeling. An osteocyte-like cell line MLO-Y4 provides a model system to examine whether gap junctions participate in the regulation of osteocyte function and signaling by mechanical stress. In this study, we show that MLO-Y4 cells are coupled and that gap junction channels mediate this coupling. Biochemical analyses show that connexin 43 (Cx43) is a major gap junction protein expressed in MLO-Y4 cells and approximately 5% of Cx43 protein is phosphorylated. MLO-Y4 cells were exposed to mechanical stress using a parallel plate flow chamber to model bone fluid flow shear stress. Fluid flow increased significantly the length of the dendritic processes, a morphological characteristic of osteocytes. A redistribution of the gap junction protein, Cx43 also was observed from a location circling the nucleus to punctate spots in the cytoplasm and in the dendritic processes. “Scrape-loading” dye transfer analyses showed that fluid flow increased intercellular coupling and increased the number of cells coupled immediately after fluid flow treatment, in direct proportion to shear stress magnitude. Although intercellular coupling continued to increase, stimulation of Cx43 protein expression during the poststress period was found to be biphasic. Cx43 protein was elevated 30 minutes after application of stress but decreased at 24 h poststress. Pulsating fluid flow had a similar stimulatory effect as steady fluid flow on gap junctions. However, this stimulatory effect in osteocyte-like cells was not observed in osteoblast-like 2T3 cells. Together, these results show that fluid flow has stimulatory effects on osteocyte-like MLO-Y4 cells with early effects on cellular morphology, opening of gap junctions, and redistribution of Cx43 protein and delayed effects on Cx43 protein expression. The high expression of Cx43 and its location in the cytoplasm also suggest that Cx43 may have unknown functions in addition to forming gap junctions. These studies indicate that gap junctions may serve as channels for signals generated by osteocytes in response to mechanical loading.
Indapamide, a Thiazide-Like Diuretic, Decreases Bone Resorption In Vitro
Authors: Lalande Agnes , Roux Sophie , Denne Marie-Annick , Stanley E. Richard , Schiavi Pierre , Guez David , De Vernejoul Marie-Christine 
Published: April 2006

Abstract
We recently showed that indapamide (IDP), a thiazide-related diuretic, increases bone mass and decreases bone resorption in spontaneously hypertensive rats supplemented with sodium. In the present study, we evaluated the in vitro effects of this diuretic on bone cells, as well as those of hydrochlorothiazide (HCTZ), the reference thiazide, and acetazolamide (AZ), a carbonic anhydrase (CA) inhibitor. We showed that 10−4 M IDP and 10−4 M AZ, as well as 10−5 M pamidronate (APD), decreased bone resorption in organ cultures and in cocultures of osteoblast-like cells and bone marrow cells in the presence of 10−8 M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We investigated the mechanism of this antiresorptive effect of IDP; IDP decreased osteoclast differentiation as the number of osteoclasts developing in coculture of marrow and osteoblast-like cells was decreased markedly. We then investigated whether IDP affected osteoblast-like cells because these cells are involved in the osteoclast differentiation. Indeed, IDP increased osteoblast-like cell proliferation and alkaline phosphatase (ALP) expression. Nevertheless, it did not modify the colony-stimulating factor 1 (CSF-1) production by these cells. In addition, osteoblast-like cells expressed the Na+/Cl− cotransporter that is necessary for the renal action of thiazide diuretics, but IDP inhibited bone resorption in mice lacking this cotransporter, so the inhibition of bone resorption and osteoclast differentiation did not involve this pathway. Thus, we hypothesized that IDP may act directly on cells of the osteoclast lineage. We observed that resorption pits produced by spleen cells cultured in the presence of soluble osteoclast differentiation factor (sODF) and CSF-1 were decreased by 10−4 M IDP as well as 10−5 M APD. In conclusion, in vitro IDP increased osteoblast proliferation and decreased bone resorption at least in part by decreasing osteoclast differentiation via a direct effect on hematopoietic precursors.
Stimulation of Protein Kinase C Activity in Cells Expressing Human Parathyroid Hormone Receptors by C- and N-Terminally Truncated Fragments of Parathyroid Hormone 1–34
Authors: Whitfield J. F. , Isaacs R. J. , Chakravarthy B. , Maclean S. , Morley P. , Willick G. , Divieti P. , Bringhurst F. R. 
Published: April 2006

Abstract
The parathyroid hormone (PTH) fragment PTH(1–34) stimulates adenylyl cyclase, phospholipase C (PLC), and protein kinase C's (PKCs) in cells that express human, opossum, or rodent type 1 PTH/PTH-related protein (PTHrP) receptors (PTHR1s). Certain carboxyl (C)-terminally truncated fragments of PTH(1–34), such as human PTH(1–31) [hPTH-(1–31)NH2], stimulate adenylyl cyclase but not PKCs in rat osteoblasts or PLC and PKCs in mouse kidney cells. The hPTH(1–31)NH2 peptide does fully stimulate PLC in HKRK B7 porcine renal epithelial cells that express 950,000 transfected hPTHR1s per cell. Amino (N)-terminally truncated fragments, such as bovine PTH(3–34) [bPTH(3–34)], hPTH(3–34)NH2, and hPTH(13–34), stimulate PKCs in Chinese hamster ovary (CHO) cells expressing transfected rat receptors, opossum kidney cells, and rat osteoblasts, but an intact N terminus is needed to stimulate PLC via human PTHR1s in HKRK B7 cells. We now report that the N-terminally truncated analogs bPTH(3–34)NH2 and hPTH(13–34)OH do activate PKC via human PTHR1s in HKRK B7 cells, although less effectively than hPTH(1–34)NH2 and hPTH(1–31)NH2. Moreover, in a homologous human cell system (normal foreskin fibroblasts), these N-terminally truncated fragments stimulate PKC activity as strongly as hPTH(1–34)NH2 and hPTH(1–31)NH2. Thus, it appears that unlike their opossum and rodent equivalents, hPTHR1s can stimulate both PLC and PKCs when activated by C-terminally truncated fragments of PTH(1–34). Furthermore, hPTHR1s, like the PTHR1s in rat osteoblasts, opossum kidney cells, and rat PTHR1-transfected CHO cells also can stimulate PKC activity by a mechanism that is independent of PLC. The efficiency with which the N-terminally truncated PTH peptides stimulate PKC activity depends on the cellular context in which the PTHR1s are expressed.
Down-Regulation of Procollagen α1[I] Messenger RNA by Titanium Particles Correlates with Nuclear Factor κB (NF-κB) Activation and Increased Rel A and NF-κB1 Binding to the Collagen Promoter
Authors: Roebuck Kenneth A. , Vermes Csaba , Carpenter Laura R. , Fritz Elizabeth A. , Narayanan Ramesh , Glant Tibor T. 
Published: April 2006

Abstract
Previously, we showed that exposure of human osteoblasts to titanium particles stimulates protein tyrosine phosphorylation (PTP), activates the transcription factor nuclear factor κB (NF-κB), and causes an approximately 50% decrease in the steady-state messenger RNA (mRNA) level of procollagen α1[I]. In this study, we identify three NF-κB binding sites within the human procollagen α1[I] gene promoter, show that titanium particles stimulate their binding of the NF-κB subunits Rel A (p65) and NF-κB1 (p50), and find NF-κB activation correlates with collagen gene suppression by titanium particles in osteoblasts. Protein tyrosine kinase (PTK) inhibitors, which significantly reduce the suppressive effect of titanium particles on collagen gene expression, inhibited NF-κB binding activity showing that titanium particle stimulation of PTK signals in osteoblasts are critical for both NF-κB activation and collagen gene expression. The antioxidant pyrrolidine dithiocarbamate (PDTC), which also inhibits the titanium particle suppression of collagen, abrogated the titanium particle activation of NF-κB, suggesting the involvement of redox signals in NF-κB-mediated collagen gene expression. The RNA polymerase II inhibitor actinomycin D (Act D) decreased procollagen α1[I] mRNA expression and effectively blocked the titanium-induced suppressive effect, suggesting that titanium particles activate a cascade of signals in osteoblasts, which result in a suppression of procollagen α1[I] mRNA. Collectively, these results show that titanium particles can activate NF-κB signaling in osteoblasts and suggest that NF-κB binding to the collagen gene promoter has a functional role in the down-regulation of procollagen α1[I] gene transcription.
1α-Hydroxyvitamin D2 Is Less Toxic but Not Bone Selective Relative to 1α-Hydroxyvitamin D3 in Ovariectomized Rats
Authors: Weber Karin , Goldberg Michel , Stangassinger Manfred , Erben Reinhold G. 
Published: April 2006

Abstract
Identification of bone selective vitamin D analogues would provide an interesting substance class for the treatment of osteoporosis. The synthetic prodrug 1α-hydroxyvitamin D2 [1α(OH)D2] has been shown to combine equal bone-preserving activity with distinctly reduced calcemic effects relative to 1α-hydroxyvitamin D3 [1α(OH)D3] in 3-month-old ovariectomized (OVX) rats. Therefore, 1α(OH)D2 may be a bone-selective compound. The aim of this study was to compare the bone protective and the calcemic activities of chronically administered 1α(OH)D2 and 1α(OH)D3 in 6-month-old OVX rats over a broad dose range from ineffective to toxic doses. Ninety-six female 6-month-old Fischer-344 rats were used for this experiment. Eighty rats were bilaterally OVX, 8 rats were sham-operated (SHAM), and 8 rats were killed at the time of surgery as a baseline control. Groups of OVX rats received vehicle alone (n = 16) or daily doses in the diet of 0.025, 0.05, 0.1, and 0.2 μg of 1α(OH)D2 or 1α(OH)D3 per kg body weight (BW) per day (n = 8 each). After calcein double-labeling, all animals were killed 3 months post-OVX. Orally administered 1α(OH)D2 was significantly less toxic compared with 1α(OH)D3 in terms of BW gain and kidney calcium content. The effects of 1α(OH)D2 and 1α(OH)D3 on serum calcium and urinary calcium excretion were generally similar at all doses in this study. Both 1α(OH)D2 and 1α(OH)D3 prevented the estrogen deficiency-induced bone loss in OVX rats, and induced profound bone anabolic effects at high dosages. 1α(OH)D3 and 1α(OH)D2 also dose-dependently increased total bone mineral density (BMD), cortical area, and cortical thickness in the tibial diaphysis of OVX rats. Bone resorption as assessed by osteoclast numbers (Oc.Ns) in vertebral cancellous bone and urinary excretion of deoxypyridinoline (DPD) was dose-dependently suppressed by 1α(OH)D2 and 1α(OH)D3. These data show that although 1α(OH)D2 was slightly but significantly less toxic compared with 1α(OH)D3, it did not have increased skeletal effects at any dose. Taken together, our findings argue against selective metabolic activation of 1α(OH)D2 in bone.
Osteopontin Deficiency Reduces Experimental Tumor Cell Metastasis to Bone and Soft Tissues
Authors: Nemoto Hiroyuki , Rittling Susan R. , Yoshitake Hiroyuki , Furuya Koichi , Amagasa Teruo , Tsuji Kunikazu , Nifuji Akira , Denhardt David T. , Noda Masaki 
Published: April 2006

Abstract
Osteopontin has been implicated in the metastasis of tumors, and human tumors with high metastatic activity often express osteopontin at high levels. Osteopontin contains an arginine-glycine-aspartate (RGD) motif that is recognized by integrin family members to promote various cell activities including attachment to substrate and it is abundant in bone, to which certain tumors preferentially metastasize. Therefore, we investigated the role of osteopontin in the experimental metastasis of tumor cells using recently established osteopontin-deficient mice. B16 melanoma cells, which produce little osteopontin, were injected into the left ventricle of osteopontin-deficient mice or wild-type mice. Animals were killed 2 weeks after injection. The number of tumors was reduced in the bones of osteopontin-deficient mice compared with the bones in wild-type mice. The number of tumors in the adrenal gland also was reduced. To investigate the osteopontin effect on metastases via a different route, we injected B16 melanoma cells into the femoral vein. Through this route, the number of lung tumors formed was higher than in the intracardiac route and was again less in osteopontin-deficient mice compared with wild-type mice. In conclusion, in an experimental metastasis assay, the number of tumors found in bone (after intracardiac injection) and lung (after left femoral vein injection) was significantly reduced in osteopontin-deficient mice compared with wild-type mice. Tumor numbers in other organs examined were small and not significantly different in the two situations.
Increased Expression of Protein Kinase Cα, Interleukin-1α, and RhoA Guanosine 5′-Triphosphatase in Osteoblasts Expressing the Ser252Trp Fibroblast Growth Factor 2 Apert Mutation: Identification by Analysis of Complementary DNA Microarray
Authors: Lomri Abderrahim , Lemonnier Jérome , Delannoy Philippe , Marie Pierre J. 
Published: April 2006

Abstract
Apert (Ap) syndrome is a craniofacial malformation characterized by premature fusion of cranial sutures (craniosynostosis). We previously showed that the Ser252Trp fibroblast growth factor receptor 2 (FGFR-2) mutation in Ap syndrome increases osteoblast differentiation and subperiosteal bone matrix formation, leading to premature calvaria ossification. In this study, we used the emerging technology of complementary DNA (cDNA) microarray to identify genes that are involved in osteoblast abnormalities induced by the Ser252Trp FGFR-2 mutation. To identify the signaling pathways involved in this syndrome, we used radioactively labeled cDNAs derived from two sources of cellular messenger RNAs (mRNAs) for hybridization: control (Co) and mutant Ap immortalized osteoblastic cells. Among genes that were differentially expressed, protein kinase Cα (PKC-α), interleukin-1α (IL-1α), and the small guanosine-5′-triphosphatase (GTPase) RhoA were increased in FGFR-2 mutant Ap cells compared with Co cells. The validity of the hybridization array was confirmed by Northern blot analysis using mRNAs derived from different cultures. Furthermore, immunochemical and Western blot analyses showed that mutant Ap cells displayed increased PKC-α, IL-1α, and RhoA protein levels compared with Co cells. Treatment of Co and Ap cells with the PKC inhibitor calphostin C decreased IL-1α and RhoA mRNA and protein levels in Ap cells, indicating that PKC is upstream of IL-1α and RhoA. Moreover, SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (MAPK), and PD-98059, a specific inhibitor of MAPK kinase (MEKK), also reduced IL-1α and RhoA expression in Ap cells. These data show that the Ser252Trp FGFR-2 mutation in Ap syndrome induces constitutive overexpression of PKC-α, IL-1α, and small GTPase RhoA, suggesting a role for these effectors in osteoblast alterations induced by the mutation. The cDNA microarray technology appears to be a useful tool to gain information on abnormal gene expression and molecular pathways induced by genetic mutations in bone cells.
Monitoring Metastatic Behavior of Human Tumor Cells in Mice with Species-Specific Polymerase Chain Reaction: Elevated Expression of Angiogenesis and Bone Resorption Stimulators by Breast Cancer in Bone Metastases
Authors: Van Der Pluijm Gabri , Sijmons Bianca , Vloedgraven Hans , Deckers Martine , Papapoulos Socrates , Löwik Clemens 
Published: April 2006

Abstract
Tumor-stroma interactions are of primary importance in determining the pathogenesis of metastasis. Here, we describe the application of sensitive competitive polymerase chain reaction (PCR) techniques for detection and quantitation of human breast cancer cells (MDA-MB-231) in an in vivo mouse model of experimental metastasis. Human-specific oligonucleotide primers in competitive PCR reactions were used to quantify the amount of MDA-MB-231 cells per tissue per organ. Using this species-specific (semi)quantitative PCR approach, gene expression patterns of (human) tumor cells or (mouse) stromal cells in metastatic lesions in the skeleton or soft tissues were investigated and compared. In all metastatic lesions, MDA-MB-231 cells express angiogenic factors (vascular endothelial growth factors [VEGFs]; VEGF-A, -B, and -C) and bone-acting cytokines (parathyroid hormone-related protein [PTHrP] and macrophage colony-stimulating factor [M-CSF]). In these metastases, PECAM-1-positive blood vessels and stromal cells of mouse origin are detected. The latter express angiogenic factors and markers of sprouting vessels (VEGF receptors flt-1/flk-1/flk-4 and CD31/PECAM-1). Strikingly, steady-state messenger RNA (mRNA) levels of VEGF-A and -B and the major bone resorption stimulators PTHrP and M-CSF by tumor cells were elevated significantly in bone versus soft tissues (p ≤ 0.05, p ≤ 0.0001, p ≤ 0.001, and p ≤ 0.05, respectively), indicating tissue-specific expression of these tumor progression factors. In conclusion, MDA-MB-231 breast cancer cells express a variety of factors in vivo that have been implicated in metastatic bone disease and that correlate with poor survival of patients with breast cancer. We hypothesize that the observed up-regulated expression of angiogenic and bone-resorbing factors by the breast cancer cells in the skeleton underlie the clinically observed osteotropism of breast cancer cells and pathogenesis of osteolytic bone metastases. The application of the species-specific competitive PCR-based assay in vivo can provide new information concerning the involvement of gene families in tumor progression and metastatic disease and greatly facilitates the study of tumor-stroma interactions in cancer invasion and metastasis.
Histomorphometry of the Embryonic Avian Growth Plate by Proton Nuclear Magnetic Resonance Microscopy
Authors: Potter Kimberlee , Landis William J. , Spencer Richard G. S. 
Published: April 2006

Abstract
Quantitative nuclear magnetic resonance (NMR) microscopy was used to characterize the biochemical and morphological properties of the different zones within the growth plate of an embryonic chick femur. For precalcified tissue, water proton transverse relaxation times (T2) and magnetization transfer values (MT) were directly and inversely dependent, respectively, on tissue cellularity, defined as the intracellular area per unit area on histological sections. T2 values extrapolated for intra- and extracellular water were 96 ms and 46 ms, respectively. The extracellular T2 was comparable with that measured for mature cartilage. The MT values extrapolated for intra- and extracellular compartments were 0.32 and 0.85, respectively. These values were comparable with those values reported in the literature for cell pellets and for mature cartilage tissue. Thus, cellularity dominated the NMR properties of this immature cartilage tissue. Mineral deposits within calcified cartilage and periosteal bone invoked NMR relaxation processes that were dependent on the inorganic mineral phase. Additionally, collagen molecules present in mineralized zones gave rise to a significant MT effect. These results show the utility of water proton NMR microscopy for assessing both the organic and inorganic phases within mineralized tissues.
Bone Mineral Density and Bone Loss Measured at the Radius to Predict the Risk of Nonspinal Osteoporotic Fracture
Authors: Gnudi Saverio , Malavolta Nazzarena , Lisi Lucia , Ripamonti Claudio 
Published: April 2006

Abstract
Low bone mineral density (BMD) and, probably, the rate of bone loss (RBL) are associated with the risk of osteoporotic fractures. To estimate the risk of nonspinal fracture in osteoporotic women, we measured BMD and RBL in a prospective study (average follow-up, 5.38 years) in 656 postmenopausal women. The women were considered in three groups: group A (whole population), group B (women under the age of 65years) and group C (women over the age of 65 years). At the beginning of the study, BMD was measured at the distal radius (DR) and at the proximal radius (PR) using a single-energy densitometer. BMD measurements made 2 years previously in the same patients were used to calculate RBL. Then patients were checked annually for nonspine fracture due to minor trauma. During follow-up, 121 nonspinal fractures were detected. Women with fractures were older and had lower BMD. With the Cox regression, age-corrected BMD at both DR and PR predicts fracture risk in groups A and B but not in group C. After correction for potential confounders, DR still predicts fractures in groups A and B whereas PR predicts fractures only in group B. In group C, only the RBL at the PR was predictive of the fracture risk as well as in the other two groups. Specific types of fractures are predictable in the whole population at the wrist. In conclusion, radial BMD predicts the risk of nonspine fractures except in women over the age of 65 years. The RBL at the PR is an effective predictor of fracture risk also in women over the age of 65 years.