bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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

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

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

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.
Characterization of Mouse Atp6i Gene, the Gene Promoter, and the Gene Expression
Authors: Deng Wenjie , Stashenko Philip , Chen Wei , Liang Yuqiong , Shimizu Ken , Li Y.-P.
Published: April 2006

Solubilization of bone mineral by osteoclasts depends on the formation of an acidic extracellular compartment through the action of a V-type ATPase. We previously cloned a gene encoding a putative osteoclast-specific proton pump subunit, termed OC-116 kDa, approved mouse Atp6i (ATPase, H+ transporting, [vacuolar proton pump] member I). The function of Atp6i as osteoclast-specific proton pump subunit was confirmed in our mouse knockout study. However, the transcription regulation of Atp6i remains largely unknown. In this study, the gene encoding mouse Atp6i and the promoter have been isolated and completely sequenced. In addition, the temporal and spatial expressions of Atp6i have been characterized. Intrachromosomal mapping studies revealed that the gene contains 20 exons and 19 introns spanning ∼11 kilobases (kb) of genomic DNA. Alignment of the mouse Atp6i gene exon sequence and predicted amino acid sequence to that of the human reveals a strong homology at both the nucleotide (82%) and the amino acid (80%) levels. Primer extension assay indicates that there is one transcription start site at 48 base pairs (bp) upstream of the initiator Met codon. Analysis of 4 kb of the putative promoter region indicates that this gene lacks canonical TATA and CAAT boxes and contains multiple putative transcription regulatory elements. Northern blot analysis of RNAs from a number of mouse tissues reveals that Atp6i is expressed predominantly in osteoclasts, and this predominant expression was confirmed by reverse-transcription polymerase chain reaction (RT-PCR) assay and immunohistochemical analysis. Whole-mount in situ hybridization shows that Atp6i expression is detected initially in the headfold region and posterior region in the somite stage of mouse embryonic development (E8.5) and becomes progressively restricted to anterior regions and the limb bud by E9.5. The expression level of Atp6i is largely reduced after E10.5. This is the first report of the characterizations of Atp6i gene, its promoter, and its gene expression patterns during mouse development. This study may provide valuable insights into the function of Atp6i, its osteoclast-selective expression, regulation, and the molecular mechanisms responsible for osteoclast activation.
Bone Strength and Related Traits in HcB/Dem Recombinant Congenic Mice
Authors: Yershov Yevgeniy , Baldini Todd H. , Villagomez Seagram , Young Todd , Martin Melissa L. , Bockman Richard S. , Peterson Margaret G. E. , Blank Robert D.
Published: April 2006

Fracture susceptibility depends jointly on bone mineral content (BMC), gross bone anatomy, and bone microarchitecture and quality. Overall, it has been estimated that 50-70% of bone strength is determined genetically. Because of the difficulty of performing studies of the genetics of bone strength in humans, we have used the HcB/Dem series of recombinant congenic (RC) mice to investigate this phenotype. We performed a comprehensive phenotypic analysis of the HcB/Dem strains including morphological analysis of long bones, measurement of ash percentage, and biomechanical testing. Body mass, ash percentage, and moment of inertia each correlated moderately but imperfectly with biomechanical performance. Several chromosome regions, on chromosomes 1, 2, 8, 10, 11, and 12, show sufficient evidence of linkage to warrant closer examination in further crosses. These studies support the view that mineral content, diaphyseal diameter, and additional nonmineral material properties contributing to overall bone strength are controlled by distinct sets of genes. Moreover, the mapping data are consistent with the existence of pleiotropic loci for bone strength-related phenotypes. These findings show the importance of factors other than mineral content in determining skeletal performance and that these factors can be dissected genetically.
Col1a1-Driven Transgenic Markers of Osteoblast Lineage Progression
Authors: Dacic S. , Kalajzic I. , Visnjic D. , Lichtler A. C. , Rowe D. W.
Published: April 2006

The modular organization of the type I collagen promoter allows creation of promoter-reporter constructs with preferential activity in different type I collagen-producing tissues that might be useful to mark cells at different stages of osteoblastic differentiation. Primary marrow stromal cell (MSC) and mouse calvarial osteoblast (mCOB) cultures were established from transgenic mice harboring different Col1a1 promoter fragments driving chloramphenicol acetyltransferase (CAT). In these models, Col1a1 messenger RNA (mRNA) and alkaline phosphatase (ALP) are the first markers of differentiation appearing soon after the colonies develop. Bone sialoprotein (BSP) is detected 2-3 days later, followed by osteocalcin (OC) expression and nodule mineralization. A 3.6 Col1a1 fragment (ColCAT3.6) initiated activity concomitant with ALP staining and type I collagen mRNA expression. In contrast, a 2.3 Col1a1 fragment (ColCAT2.3) became active coincident with BSP expression. The pattern of transgene expression assessed by immunostaining was distinctly different. ColCAT3.6 was expressed within and at the periphery of developing nodules whereas the ColCAT2.3 expression was restricted to the differentiated nodules. The feasibility of using green fluorescent protein (GFP) as a marker of osteoblast differentiation was evaluated in ROS17/2.8 cells. A 2.3-kilobase (kb) Col1a1 promoter driving GFP (pOB4Col2.3GLP) was stably transfected into the cell line and positive clones were selected. Subcultures lost and then regained GFP expression that was localized in small clusters of cells throughout the culture. This suggests that expression from the 2.3-kb Col1A1 fragment is determined by the state of differentiation of the ROS17/2.8 cells. Col1a1 transgenes should be useful in appreciating the heterogeneity of a primary or immortalized culture undergoing osteoblastic differentiation.
Continuous Parathyroid Hormone and Estrogen Administration Increases Vertebral Cancellous Bone Volume and Cortical Width in the Estrogen-Deficient Rat
Authors: Zhou H. , Shen V. , Dempster D. W. , Lindsay R.
Published: April 2006

Generally, it is believed that intermittent administration of parathyroid hormone (PTH) has an anabolic effect on the skeleton, whereas continuous administration is catabolic. However, there is evidence that continuous exposure to PTH may have an anabolic effect, for example, in patients with mild primary hyperparathyroidism (PHPT). The possibility of delivering PTH continuously may have important implications for the treatment of osteoporosis. Furthermore, estrogen treatment may be useful in the medical management of PHPT. Therefore, we examined the skeletal effects of continuous administration of PTH, with or without estrogen, in the estrogen-deficient rat with established osteopenia. Forty 7-month-old SD rats were divided into four ovariectomy (OVX) groups and one sham-operated group. Eight weeks post-OVX, three groups received subcutaneous implants of Alzet mini pumps loaded with PTH(1-34) (30 μg/kg per day), 17β-estradiol (10 μg/kg per day) pellet, or both PTH and 17β-estradiol separately for 4 weeks. OVX and sham control groups were given the mini pumps loaded with vehicle. Two doses of calcein (10 mg/kg) were given subcutaneously to all rats 2 days and 8 days before death. Histomorphometry was performed on cancellous and cortical bone of the fourth lumbar vertebra. At 3 months, post-OVX rats displayed bone loss with high bone turnover. Estrogen reversed OVX-mediated high turnover without restoring cancellous bone volume (BV/TV). PTH infusion further increased bone turnover and partially restored BV/TV. However, PTH infusion increased cortical porosity. Estrogen inhibited PTH-mediated cancellous bone resorption and substantially increased BV/TV above sham control. The combined treatment was associated with a significant increase in peritrabecular fibrosis and woven bone formation. The combined treatment of PTH infusion and estrogen replacement enhanced cortical width but estrogen did not prevent the PTH-induced cortical tunneling. We conclude that continuous administration of PTH and estrogen increases cortical porosity but has substantial beneficial effects on vertebral cancellous bone volume and cortical width in OVX rats.
Ultrasonic Backscatter and Transmission Parameters at the Os Calcis in Postmenopausal Osteoporosis
Authors: Roux Christian , Roberjot Valéry , Porcher Raphaël , Kolta Sami , Dougados Maxime , Laugier Pascal
Published: April 2006

Ultrasound technology has emerged as a new tool in the assessment of osteoporosis. Ultrasound parameters usually are measured in transmission; there is a potential for the analysis of backscattered signals to provide information on bone microarchitecture. The aim of this study was to explore a new technological development of the method, adding backscatter coefficient to transmission parameters, and to examine the appropriate thresholds to identify postmenopausal osteoporotic women. We examined 210 postmenopausal women (including 60 with osteoporotic fractures) and 30 healthy premenopausal controls. They had lumbar spine and hip bone mineral density (BMD) measurement and quantitative ultrasound (QUS) evaluation at the os calcis, measured in transmission (broadband ultrasound attenuation [BUA], speed of sound [SOS], ratio of transit time [dt] to BUA [dt/BUA], and “strength” index [STI]) and reflexion (broadband ultrasound backscattering [BUB]). The standardized CVs (sCVs) were between 2.27% and 3.40% for QUS measured in transmission and 4.41% for BUB. The odds ratio (OR) for fracture discrimination adjusted for age was 2.77 for hip BMD and between 1.6 and 2.9 for QUS. After adjustment for hip BMD, ORs were still highly significant for SOS, STI, and dt/BUA. According to hip BMD T score, prevalence of osteoporosis in our population was 39%. To detect the same prevalence, T scores ranged between −0.95 and −1.42 for QUS. QUS parameters have adequate ability to discriminate osteoporotic patients from controls. The World Health Organization (WHO) threshold for diagnosis of osteoporosis does not apply to this technology. The clinical utility of BUB at the os calcis, in addition to usual ultrasound parameters, is not yet proven. However, BUB evaluation, which does not require two transducers and may be implemented in conventional reflection mode systems, warrants further studies.
Colocalization of Glucocorticoid and Mineralocorticoid Receptors in Human Bone
Authors: Beavan Siân , Horner Alan , Bord Sharyn , Ireland Deborah , Compston Juliet
Published: April 2006

Osteoporosis is a poorly understood but common complication of glucocorticoid therapy. The actions of glucocorticoids are mediated via glucocorticoid receptors (GRs), but in vitro, glucocorticoids also can bind to mineralocorticoid receptors (MRs). It is not known if MR protein is present in human bone and little is known of GR isoform expression (GRα and GRβ). GR and MR protein expression and possible sites of action were investigated in neonatal rib and adult iliac crest biopsy specimens using antibodies specific for MR, GRα, and GRαβ. Colocalization [MR GRα] [MR GRαβ] was performed using fluorescent-conjugated secondary antibodies. GRα, GRβ, and MR show distinct but overlapping patterns of expression, suggesting important functions for each receptor type. Osteoclasts showed no staining for GRα but strong staining for GRαβ, indicating expression of GRβ and a specific role in addition to antagonizing the transcriptional activity of GRα. MR also was observed in osteoclasts and colocalized with GRαβ. Coexpression of MR, GRα, and GRαβ was seen in osteoblasts. Reverse-transcription-polymerase chain reaction (RT-PCR) of cultured osteoblast RNA confirmed expression of both GRα and GRβ. Osteocytes stained with MR, GRα, and GRαβ antibodies but to a lesser degree than osteoblasts. In the neonatal rib cartilage, staining for GRα, GRαβ, and MR was present in approximately one-half of the resting and hypertrophic chondrocytes and in most of proliferating chondrocytes and chondrocytes within the mineralizing matrix. Identification of MR raises the possibility that the physiological and pharmacologic effects of glucocorticoids on bone may be mediated via MR as well as GR and that GRα, GRβ, and MR synergize to influence corticosteroid metabolism in human bone.
Digital Topological Analysis of In Vivo Magnetic Resonance Microimages of Trabecular Bone Reveals Structural Implications of Osteoporosis
Authors: Wehrli Felix W. , Gomberg Bryon R. , Saha Punam K. , Song Hee Kwon , Hwang Scott N. , Snyder Peter J.
Published: April 2006

Osteoporosis is a disease characterized by bone volume loss and architectural deterioration. The majority of work aimed at evaluating the structural implications of the disease has been performed based on stereologic analysis of histomorphometric sections. Only recently noninvasive imaging methods have emerged that provide sufficient resolution to resolve individual trabeculae. In this article, we apply digital topological analysis (DTA) to magnetic resonance microimages (μ-MRI) of the radius obtained at 137 × 137 × 350 μm3 voxel size in a cohort of 79 women of widely varying bone mineral density (BMD) and vertebral deformity status. DTA is a new method that allows unambiguous determination of the three-dimensional (3D) topology of each voxel in a trabecular bone network. The analysis involves generation of a bone volume fraction map, which is subjected to subvoxel processing to alleviate partial volume blurring, followed by thresholding and skeletonization. The skeletonized images contain only surfaces, profiles, curves, and their mutual junctions as the remnants of trabecular plates and rods after skeletonization. DTA parameters were compared with integral BMD in the lumbar spine and femur as well as MR-derived bone volume fraction (BV/TV). Vertebral deformities were determined based on sagittal MRIs of the spine with a semiautomatic method and the number of deformities counted after threshold setting. DTA structural indices were found the strongest discriminators of subjects with deformities from those without deformities. Subjects with deformities (n = 29) had lower topological surface (SURF) density (p < 0.0005) and surface-to-curve ratio (SCR; a measure of the ratio of platelike to rodlike trabeculae; p < 0.0005) than those without. Profile interior (PI) density, a measure of intact trabecular rods, was also lower in the deformity group (p < 0.0001). These data provide the first in vivo evidence for the structural implications inherent in postmenopausal osteoporosis accompanying bone loss, that is, the conversion of trabecular plates to rods and disruption of rods due to repeated osteoclastic resorption.
Influence of the Upper Femur and Pelvic Geometry on the Risk and Type of Hip Fractures
Authors: Partanen Juha , Jämsä Timo , Jalovaara Pekka
Published: April 2006

The geometry of the upper femur has been reported to associate with the hip fracture risk in postmenopausal women. However, these associations seem to be partly conflicting, probably because of differences in measurement setup. Here, we compared the upper femur and pelvic geometries of 70 hip fracture patients (46 cervical and 24 trochanteric fractures) and 40 age-adjusted controls based on plain anteroposterior radiographs, eliminating the possible sources of inaccuracy as far as possible by using a standardized patient position and calibrated dimension measurements by digital image analysis. The femoral neck/shaft angle (NSA) was larger in the fracture patients compared with the controls (p < 0.001). The fracture group had thinner cortices in the upper femur than the controls (p < 0.001). The femoral shaft diameter (FSD; p < 0.001), trochanter width (TW; p < 0.01), and the pelvic dimensions, that is, the smallest outer pelvic diameter (SOPD; p < 0.01) and the largest inner pelvic diameter (LIPD; p < 0.05) were smaller in the fracture group. Comparing the fracture types, we found NSA larger in the cervical hip fracture patients than in the patients with a trochanteric fracture (p < 0.01). The femoral neck/shaft cortex ratio was lower and the FSD was smaller in the cervical hip fracture group (p < 0.05). Acetabular width (AW) was greater and the SOPD was wider in the cervical fracture patients (p < 0.01). We concluded that the upper femur and pelvic dimensions as defined from calibrated and position-standardized plain radiographs are useful in the evaluation of hip fracture risk and fracture type.
Large-Scale Screening for Candidate Genes of Ossification of the Posterior Longitudinal Ligament of the Spine
Authors: Furushima Kozo , Shimo-Onoda Kazuki , Maeda Shingo , Nobukuni Takahiro , Ikari Katsunori , Koga Hiroaki , Komiya Setsuro , Nakajima Toshiaki , Harata Seiko , Inoue Ituro
Published: April 2006

Ossification of the posterior longitudinal ligament of the spine (OPLL) is the predominant myelopathy among Japanese, and is usually diagnosed by ectopic bone formation in the paravertebral ligament in Japanese and other Asians. To detect genetic determinants associated with OPLL, we performed an extensive nonparametric linkage study with 126 affected sib-pairs using markers for various candidate genes by distinct analyses, SIBPAL and GENEHUNTER. Eighty-eight candidate genes were selected by comparing the genes identified by complementary DNA (cDNA) microarray analysis of systematic gene expression profiles during osteoblastic differentiation of human mesenchymal stem cells with the genes known to be involved in bone metabolism. Of the 24 genes regulated during osteoblastic differentiation, only one, the alpha B crystalline gene, showed evidence of linkage (p = 0.016, nonparametric linkage [NPL] score = 1.83). Of 64 genes known to be associated with bone metabolism, 7 showed weak evidence of linkage by SIBPAL analysis (p < 0.05): cadherin 13 (CDH13), bone morphogenetic protein 4 (BMP4), proteoglycan 1 (PRG1), transforming growth factor beta 3 (TGFb3), osteopontin (OPN), parathyroid hormone receptor 1 (PTHR1), and insulin-like growth factor 1 (IGF1). Among these genes, BMP4 (NPL = 2.23), CDH13 (NPL = 2.00), TGFb3 (NPL = 1.30), OPN (NPL = 1.15), and PTHR1 (NPL = 1.00) showed evidence of linkage by GENEHUNTER. Only BMP4 reached criteria of suggestive evidence of linkage. Because this gene is a well-known factor in osteogenetic function, BMP4 should be screened in further study for the polymorphism responsible.
Use of Type I Collagen Green Fluorescent Protein Transgenes to Identify Subpopulations of Cells at Different Stages of the Osteoblast Lineage
Authors: Kalajzic I. , Kalajzic Z. , Kaliterna M. , Gronowicz G. , Clark S. H. , Lichtler A. C. , Rowe D.
Published: April 2006

Green fluorescent protein (GFP)-expressing transgenic mice were produced containing a 3.6-kilobase (kb; pOBCol3.6GFPtpz) and a 2.3-kb (pOBCol2.3GFPemd) rat type I collagen (Col1a1) promoter fragment. The 3.6-kb promoter directed strong expression of GFP messenger RNA (mRNA) to bone and isolated tail tendon and lower expression in nonosseous tissues. The 2.3-kb promoter expressed the GFP mRNA in the bone and tail tendon with no detectable mRNA elsewhere. The pattern of fluorescence was evaluated in differentiating calvarial cell (mouse calvarial osteoblast cell [mCOB]) and in marrow stromal cell (MSC) cultures derived from the transgenic mice. The pOBCol3.6GFPtpz-positive cells first appeared in spindle-shaped cells before nodule formation and continued to show a strong signal in cells associated with bone nodules. pOBCol2.3GFPemd fluorescence first appeared in nodules undergoing mineralization. Histological analysis showed weaker pOBCol3.6GFPtpz-positive fibroblastic cells in the periosteal layer and strongly positive osteoblastic cells lining endosteal and trabecular surfaces. In contrast, a pOBCol2.3GFPemd signal was limited to osteoblasts and osteocytes without detectable signal in periosteal fibroblasts. These findings suggest that Col1a1GFP transgenes are marking different subpopulations of cells during differentiation of skeletal osteoprogenitors. With the use of other promoters and color isomers of GFP, it should be possible to develop experimental protocols that can reflect the heterogeneity of cell differentiation in intact bone. In primary culture, this approach will afford isolation of subpopulations of these cells for molecular and cellular analysis.
Bone Strength at Clinically Relevant Sites Displays Substantial Heterogeneity and Is Best Predicted From Site-Specific Bone Densitometry
Authors: Eckstein Felix , Lochmüller Eva-Maria , Lill Christoph A. , Kuhn Volker , Schneider Erich , Delling Günter , Müller Ralph
Published: April 2006

In this study we test the hypotheses that mechanical bone strength in elderly individuals displays substantial heterogeneity among clinically relevant skeletal sites, that ex situ dual-energy X-ray absorptiometry (DXA) provides better estimates of bone strength than in situ DXA, but that a site-specific approach of bone densitometry is nevertheless superior for optimal prediction of bone failure under in situ conditions. DXA measurements were obtained of the lumbar spine, the left femur, the left radius, and the total body in 110 human cadavers (age, 80.6 ± 10.5 years; 72 female, 38 male), including the skin and soft tissues. The bones were then excised, spinal and femoral DXA being repeated ex situ. Mechanical failure tests were performed on thoracic vertebra 10 and lumbar vertebra 3 (compressive loading of a functional unit), the left and right femur (side impact and vertical loading configuration), and the left and right distal radius (fall configuration, axial compression, and 3-point-bending). The failure loads displayed only very moderate correlation among sites (r = 0.39 to 0.63). Ex situ DXA displayed slightly higher correlations with failure loads compared with those of in situ DXA, but the differences were not significant and relatively small. Under in situ conditions, DXA predicted 50-60% of the variability in bone failure loads at identical (or closely adjacent) sites, but only around 20-35% at distant sites, advocating a site-specific approach of densitometry. These data suggest that mechanical competence in the elderly is governed by strong regional variation, and that its loss in osteoporosis may not represent a strictly systemic process.
Antiangiogenic Treatment Delays Chondrocyte Maturation and Bone Formation During Limb Skeletogenesis
Authors: Yin Melinda , Gentili Chiara , Koyama Eiki , Zasloff Michael , Pacifici Maurizio
Published: April 2006

Hypertrophic chondrocytes have important roles in promoting invasion of cartilage by blood vessels and its replacement with bone. However, it is unclear whether blood vessels exert reciprocal positive influences on chondrocyte maturation and function. Therefore, we implanted beads containing the antiangiogenic molecule squalamine around humeral anlagen in chick embryo wing buds and monitored the effects over time. Fluorescence microscopy showed that the drug diffused from the beads and accumulated in humeral perichondrial tissues, indicating that these tissues were the predominant targets of drug action. Diaphyseal chondrocyte maturation was indeed delayed in squalamine-treated humeri, as indicated by reduced cell hypertrophy and expression of type X collagen, transferrin, and Indian hedgehog (Ihh). Although reduced in amount, Ihh maintained a striking distribution in treated and control humeri, being associated with diaphyseal chondrocytes as well as inner perichondrial layer. These decreases were accompanied by lack of cartilage invasion and tartrate-resistant acid phosphatase-positive (TRAP+) cells and a significant longitudinal growth retardation. Recovery occurred at later developmental times, when in fact expression in treated humeri of markers such as matrix metalloproteinase 9 (MMP-9) and connective tissue growth factor (CTGF) appeared to exceed that in controls. Treating primary cultures of hypertrophic chondrocytes and osteoblasts with squalamine revealed no obvious changes in cell phenotype. These data provide evidence that perichondrial tissues and blood vessels in particular influence chondrocyte maturation in a positive manner and may cooperate with hypertrophic chondrocytes in dictating the normal pace and location of the transition from cartilage to bone.
L-Type Calcium Channels Mediate Mechanically Induced Bone Formation In Vivo
Authors: Li Jiliang , Duncan Randall L. , Burr David B. , Turner Charles H.
Published: April 2006

Cell and tissue culture studies suggest that the long-lasting (L-type) voltage-sensitive calcium channels (VSCC) play a role in the signaling cascade in bone cells after mechanical loading. We investigated whether the L-type VSCC mediates mechanically induced bone formation in vivo using two L-type VSCC antagonists verapamil and nifedipine. Female Sprague-Dawley rats were divided into five groups: control group (Veh), two verapamil-treated groups (20 mg/kg, Vera-L; 100 mg/kg, Vera-H), and two nifedipine-treated groups (20 mg/kg, Nife-L; 100 mg/kg, Nife-H). One bout of mechanical loading was applied to the right tibia 90 minutes after oral administration of verapamil or 30 minutes after oral administration of nifedipine. Mechanical loading increased mineralizing surface (MS/bone surface [BS]), mineral apposition rate (MAR), and bone formation rate (BFR/BS) on the endocortical surface in loaded tibias of control animals compared with nonloaded (left) tibias. Verapamil and nifedipine suppressed the load-induced increase in BFR/BS observed in vehicle-treated controls by 56–61% (p < 0.01) and 56–74% (p < 0.01), respectively. Yet, significant differences in MS/BS and BFR/BS between right and left limbs were found in verapamil- and nifedipine-treated animals, indicating that the treatments did not completely abolish load-induced bone formation. This study shows that blocking the L-type calcium channel in vivo substantially suppresses the mechanically induced increase in bone formation that normally would occur and suggests that the L-type calcium channel mediates mechanically induced bone adaptation in vivo.
Dentin Matrix Protein 1, a Target Molecule for Cbfa1 in Bone, Is a Unique Bone Marker Gene
Authors: Feng Jian Q. , Zhang Jianghong , Dallas Sarah L. , Lu Yongbo , Chen Shuo , Tan Xiaoyu , Owen Michael , Harris Stephen E. , Macdougall Mary
Published: April 2006

Dentin matrix protein 1 (Dmp1), a phosphoprotein highly linked to dentin formation, has also been reported to be expressed in the skeleton. However, the role of Dmp1 in skeletal tissues remains unclear. To clarify the role of Dmp1 in bone formation, we characterized the expression profile of Dmp1 in bone and cartilage and examined whether Dmp1 expression was regulated by core-binding factor a1 (Cbfa1). Studies of fetal rat calvarial (FRC) cell cultures showed that the expression of Dmp1 was associated closely with “bone nodule” formation and mineralization in vitro. In situ hybridization studies were performed to examine the spatial and temporal expression patterns of Dmp1 during development in mouse embryos from 12.5 day postcoitus (dpc) to 8 weeks postnatal; these studies showed that Dmp1 first appeared in hypertrophic cartilage cells, followed by osteoblasts, and later was expressed strongly in osteocytes. The expression profiles of Cbfa1 and Dmp1 overlapped in both cartilage and bone during development, with Cbfa1 preceding Dmp1. Examination of Dmp1 expression in Cbfa1−/− mice revealed that Dmp1 was absent in the developing bones of Cbfa1-null mice, whereas there was essentially no change in Dmp1 expression in the arrested tooth bud. Transient transfection studies showed forced expression of Dmp1 under the control of Cbfa1 and gel shift data indicated the presence of a functional osteocalcin-specific element (OSE)-2 response element in the Dmp1 proximal promoter region. However, in vitro promoter studies suggested that regulation of Dmp1 by Cbfa1 was not mediated by direct binding of Cbfa1 to this site and may be through indirect mechanisms. These studies highlight Dmp1 as a unique marker gene for osteoblastic differentiation. The close association of Dmp1 and Cbfa1 in the developing skeleton suggests that Dmp1 may play an important role in bone formation.
Involvement of Cyclic Adenosine Monophosphate-Dependent Protein Kinase A and Pertussis Toxin-Sensitive G Proteins in the Migratory Response of Human CD14+ Mononuclear Cells to Katacalcin
Authors: Kaneider Nicole C. , Egger Petra , Wiedermann Franz J. , Ritter Markus , Wöll Ewald , Wiedermann Christian J.
Published: April 2006

Katacalcin (KC) belongs to a small family of polypeptides that are encoded by the calc-1 gene and also include calcitonin (CT) and procalcitonin NH2-terminal cleavage peptide (N-ProCT). Biological roles of KC or N-ProCT are unknown. To determine whether these polypeptides affect leukocyte function, forearm venous blood polymorphonuclear neutrophils and CD14+ peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors. Cell migration was assessed in a blindwell chemotaxis chamber using nitrocellulose micropore filters. Cellular levels of cyclic adenosine monophosphate (cAMP) were measured by HPLC; activation of protein kinase A was studied by Western blot. Fluorochrome-labeled peptide binding to cells was studied by fluorescence-activated cell sorting (FACS) and intracellular calcium transients were studied by confocal microscopy with FLUO-3. KC elicited concentration-dependent migration of CD14+ PBMC at concentrations from the atomolar to the micromolar range and deactivated attractant-induced chemotaxis. CT N-terminal flanking peptide had no such effect. Neutrophils did not migrate toward any of those peptides and their oxygen-free radical release was not affected as measured fluorometrically. Functional responses of CD14+ PBMC to KC correlated to forskolin-sensitive cAMP accumulation in cells and were inhibited by protein kinase A inhibitor (PKI) and Rp diastereomer of adenosine 3′,5′-cyclic monophosphorothioate. Treatment of CD14+ PBMC with KC activated protein kinase ACα. Intracellular calcium was decreased with CT, KC, and procalcitonin (PCT). Binding studies showed that KC might share the binding site with CT and PCT. Data indicate that KC regulates human CD14+ PBMC migration via signaling events involving protein kinase A-dependent cAMP pathways.
Three-Dimensional-Line Skeleton Graph Analysis of High-Resolution Magnetic Resonance Images: A Validation Study From 34-μm-Resolution Microcomputed Tomography
Authors: Pothuaud Laurent , Laib Andres , Levitz Pierre , Benhamou Claude L. , Majumdar Sharmila
Published: April 2006

The resolution achievable in vivo by magnetic resonance imaging (MRI) techniques is not sufficient to depict precisely individual trabeculae and, thus, does not permit the quantification of the “true” trabecular bone morphology and topology. Nevertheless, the characterization of the “apparent” trabecular bone network derived from high-resolution MR images (MRIs) and their potential to provide information in addition to bone mineral density (BMD) alone has been established in studies of osteoporosis. The aim of this work was to show the ability of the three-dimensional-line skeleton graph analysis (3D-LSGA) to characterize high-resolution MRIs of trabecular bone structure. Fifteen trabecular bone samples of the distal radius were imaged using the high-resolution MRI (156 × 156 × 300 μm3) and microcomputed tomography (μCT; 34 × 34 × 34 μm3). After thresholding, the 3D skeleton graph of each binary image was obtained. To remove the assimilated-noise branches of the skeleton graph and smooth this skeleton graph before it was analyzed, we defined a smoothing length criterion (lc), such that all “termini” branches having a length lower than lc were removed. Local topological and morphological LSGA measurements were performed from MRIs and μCT images of the same samples. The correlations between these two sets of measurements were dependent on the smoothing criterion lc, reaching R2 = 0.85 for topological measurements and R2 = 0.57–0.64 for morphological measurements. 3D-LSGA technique could be applied to in vivo high-resolution MRIs of trabecular bone structure, giving an indirect characterization of the microtrabecular bone network.
Role of the Bone Marrow Microenvironment in Multiple Myeloma
Authors: Roodman G. David
Published: April 2006

On June 26–27, 2001, the Sixth Research Roundtable in Multiple Myeloma, entitled “The Role of the Bone Microenvironment in Multiple Myeloma,” was held and focused on the biology of cell-to-cell interactions, the mediators of bone disease, and novel treatment strategies for myeloma. Studies on cell-cell interactions showed that vascular cell adhesion molecule 1, expressed by local endothelial and stromal cells, binds to tumor cell surface integrins in which expression may be increased by tumor cell-derived chemokines such as macrophage inflammatory protein (MIP) 1α. These adhesive interactions increase production and release of vascular endothelial growth factor (VEGF). Studies on myeloma bone disease showed the ligand for receptor activator of nuclear transcription factor-κB (RANKL) was expressed on tumor cells and stromal cells associated with myeloma cells and was critical for osteoclast-induced osteolysis. Blockade of RANKL suppressed osteoclast maturation, bone resorption, and tumor development. Bisphosphonates, in addition to reducing osteoclast mobility and inducing osteoclast apoptosis, also decreased tumor cell adhesion to stroma. Immunomodulatory drugs such as thalidomide analogues targeted these tumor cell-stromal cell interactions, blocking both secretion of cytokines and activation of intracellular signaling pathways required for tumor survival and growth. These agents induced tumor cell apoptosis, decreased neovascularization, and potentiated natural killer cell activity. The proteasome inhibitor PS-341 also prevented expression of adhesion molecules and cytokines and triggered tumor cell apoptosis, even in drug-resistant cell lines, while showing minimal activity in healthy cells. In addition, potential therapeutic agents under investigation, which included RANKL antagonists, protein prenylation inhibitors, and osteoblast growth factors, were discussed.
Bone Mineralization in Polyostotic Fibrous Dysplasia: Histomorphometric Analysis
Authors: Terpstra Leonieke , Rauch Frank , Plotkin Horacio , Travers Rose , Glorieux Francis H.
Published: April 2006

Fibrous dysplasia (FD) of bone can be complicated by renal phosphate wasting. The effect of hypophosphatemia on normal and dysplastic bone of FD patients has not been well characterized. In this study, we compared serum phosphorus (sPi) levels to histomorphometric findings in 27 iliac bone samples from 23 children and adolescents (aged 4.2–16.4 years) with polyostotic FD. The samples were separated into two groups, based on the presence (n = 10) or absence (n = 17) of a dysplastic lesion within the specimen. Histomorphometric results were compared with those from 18 age-matched control subjects without metabolic bone disease. In dysplastic lesions, trabeculae were clearly thinner and increased in number. Osteoid indices, osteoblast surface per bone surface, and mineralization lag time were elevated in dysplastic areas, but there was no detectable effect of sPi concentrations on these indices. In nondysplastic bone tissue, low sPi levels were associated with mildly increased osteoid thickness and prolonged mineralization lag time. None of the 13 patients in whom hand X-rays were available at the time of biopsy had radiological signs of rickets. In conclusion, low sPi can cause a mild systemic mineralization defect in FD, but the more severe mineralization defect seen in dysplastic lesions is independent of sPi levels. It is debatable whether the mild systemic mineralization defect warrants treatment with oral phosphorus supplementation if signs of rickets are absent.
Greatly Increased Cancellous Bone Formation With Rapid Improvements in Bone Structure in the Rat Maternal Skeleton After Lactation
Authors: Bowman B. M. , Siska C. C. , Miller S. C.
Published: April 2006

There is a decrease in cancellous bone mass and strength during lactation but these are partially or completely reconstituted in the postlactational period. The purpose of this study was to determine changes in cancellous bone structure and formation after lactation in established breeder rats. For this, rats were taken at the end of the second pregnancy (Preg-2) and second lactation (Lac-2) and 2, 4, and 6 weeks after weaning. Nulliparous (NP) groups were included for comparisons. Bone structure was measured using morphometric methods and bone dynamics by histomorphometry. Tibial metaphyseal cancellous bone was lost during the first reproductive cycle, as expected, and again depleted during the Lac-2. Bone formation indices were elevated at the end of Lac-2, compared with those at the end of the second pregnancy or in the nulliparous animals. Within 2 weeks after the second weaning, the amount of double-labeled surface (dLS) increased ∼800%, the mineralizing surface (MS) increased >400% with similar increases in bone formation rates (BFRs), compared with already elevated bone formation measured at the end of Lac-2. From 2 to 4 weeks after lactation, there were commensurate increases in cancellous bone mass and structural indices with essentially complete restoration of cancellous bone volume and structure compared with that measured at the end of Preg-2. The results show rapid and substantial increases in bone formation with reconstitution of cancellous bone mass and structure after lactation in rats. The skeletal changes that occur during the postlactational period may serve to prepare and protect the maternal skeleton for subsequent reproductive cycles.