bluuebunny/crossref_metadata_2025 huggingface dataset:
Collagenase-3 (MMP-13) and Integral Membrane Protein 2a (Itm2a) are Marker Genes of Chondrogenic/Osteoblastic Cells in Bone Formation: Sequential Temporal, and Spatial Expression of Itm2a, Alkaline Phosphatase, MMP-13, and Osteocalcin in the Mouse
Authors: Tuckermann Jan P. , Pittois Karen , Partridge Nicola C. , Merregaert Joseph , Angel Peter
Published: April 2006

Endochondral bone formation requires the action of cells of the chondrocytic and osteoblastic lineage, which undergo continuous differentiation during this process. To identify subpopulations of resting, proliferating, and hypertrophic chondrocytes and osteoblasts involved in bone formation, we have identified here two novel marker genes present in endochondral and intramembranous ossification. Using Northern blot analysis and in situ hybridization on parallel sections of murine embryos and bones of newborn mice we compared the expression pattern of the recently cloned Itm2a and MMP-13 (collagenase-3) genes with that of established marker genes for bone formation, such as alkaline phosphatase (ALP), osteocalcin (OC), and collagen type X, during endochondral and intramembranous ossification. During embryonic development expression of Itm2a and ALP was detectable at midgestation (11.5 days postcoitum [dpc]) and increased up to 16.5 dpc. MMP-13 and OC expression started at 14.5 dpc and 16.5 dpc, respectively. This temporal expression was reflected in the spatial distribution of these markers in the growth plate of long bones. In areas undergoing endochondral ossification Itm2a expression was found in chondrocytes of the resting and the proliferating zones. Expression of ALP and MMP-13 are mutually exclusive: ALP transcripts were found only in collagen type X positive hypertrophic chondrocytes of the upper zone. MMP-13 expression was restricted to chondrocytes of the lower zone of hypertrophic cartilage also expressing collagen type X. In osteoblasts involved in endochondral and intramembranous ossification Itm2a was not present. ALP, MMP-13, and OC were mutually exclusively expressed in these cells suggesting a differentiation-dependent sequential expression of ALP, MMP-13, and OC. The identification of the continuum of sequential expression of Itm2a, ALP, MMP-13, and OC will now allow us to establish a series of marker genes that are highly suitable to characterize bone cells during chondrocytic and osteoblastic differentiation in vivo.
Cellular Localization of Neprilysin in Mouse Bone Tissue and Putative Role in Hydrolysis of Osteogenic Peptides
Authors: Ruchon Andréa Frota , Marcinkiewicz Mieczyslaw , Ellefsen Kim , Basak Ajoy , Aubin Jane , Crine Philippe , Boileau Guy
Published: April 2006

The regulation of osteoblast and osteoclast metabolism is mediated by both hormones and local bone peptide factors. Peptides and hormones are under control of membrane peptidases such as Neprilysin (NEP). NEP is a widely distributed cell-surface zinc-metallopeptidase that is involved in the regulation of several important physiological processes by controlling the half-life of bioactive peptides. Although NEP is known to be present in skeletal tissues, neither its cellular localization nor its function have been established. To address this question, we examined NEP distribution in bones of postnatal mouse. In situ hybridization (ISH) and immunohistochemistry showed that NEP messenger RNA (mRNA) and protein are associated with bone-forming cells including presumptive osteoblast precursors, preosteoblasts, osteoblasts, and osteocytes. NEP levels in newborn and adult mice bones also were compared by immunoblotting. Higher amounts of NEP immunoreactivity were observed in newborn as compared with adult bones, suggesting a relationship between NEP expression and bone growth. To further explore this hypothesis, we monitored in vitro NEP proteolytic activity using a series of synthetic osteogenic peptides such as parathyroid hormone-related peptide 1–43 (PTHrP1–34), osteostatin (PTHrP107–139), osteogenic growth peptide (OGP), calcitonin, α-calcitonin gene-related peptide (α-CGRP), and PTH1–34. Except for PTH1–34, all peptides were found to be NEP substrates.
Estrogen Agonist (Zeranol) Treatment in a Castrated Male Lamb Model: Effects on Growth and Bone Mineral Accretion
Authors: Chanetsa F. , Dr. Hillman L. S. , Thomas M. G. , Keisler D. H.
Published: April 2006

The mechanism of estrogen's action on bone mineralization in children has received little attention. Our objective was to determine the effect of time (developmentally) and duration of exposure to an estrogen agonist (zeranol) on bone growth and mineralization using a castrated male lamb model. At birth, 40 male lambs were castrated and within 14 days of birth (day = 0) they were assigned (n = 10 per group) to age-matched control lambs (C-AGE) or to receive a 12.5-mg zeranol implant as follows: E-0, implanted on days 0, 45, 90, and 135; E-90, implanted on days 90 and 135; and E-0,90, implanted on days 0, 90, and 135. Lambs were studied for 163 days. Serum was collected on days 28, 73, 118, 135, and 163 and analyzed for minerals (Ca, P, and Mg), markers of bone remodeling (bone alkaline phosphatase [ALP] and tartrate resistant acid phosphatase [TRAP]), 1,25-dihydroxyvitamin D [1,25(OH)2D], growth hormone (GH), and insulin-like growth factor I (IGF-I). Whole-body bone mineral content (BMC), bone mineral density (BMD), fat mass, and lean mass were determined by dual energy X-ray absorptiometry (DEXA) on days 28, 73, 118, and 163. There was a linear increase in growth at all time points. Whole-body BMC, weight, and lean mass of C-AGE and E-90 lambs were less than E-0, and E-0,90 lambs at all time points. Whole-body BMD of C-AGE and E-90 lambs was less than E-0 and E-0,90 lambs at 28 days and 73 days; however, after implantation at day 90 whole-body BMD of E-90 lambs was similar to E-0 and E-0,90 lambs at day 118 and day 163 and all three were greater than C-AGE lambs. There was no effect of treatment on calcium absorption, serum minerals, hormones, or markers of bone remodeling. We conclude from these data that treatment of growing castrated lambs with an estrogen agonist from birth augments growth, whereas delaying estrogen agonist treatment does not facilitate growth but appears to augment bone mineral accretion. We suggest these observations may have clinical relevance, and deserve consideration when treating children with delays in growth and bone mineral accretion.
Bone Structure in Patients with Low Bone Mineral Density With or Without Vertebral Fractures
Authors: Oleksik Anna , Ott Susan M. , Vedi Shobna , Bravenboer Nathalie , Compston Juliet , Lips Paul
Published: April 2006

Vertebral fractures (VFX) are caused by low bone mass and microstructural deterioration of bone tissue. The latter is not well defined. We investigated bone structure in transiliac biopsy specimens from 88 volunteers. Biopsy specimens were obtained at baseline in the Multiple Outcomes of Raloxifene Evaluation trial, a prospective study in osteoporotic (BMD ≤ −2.5 T score) postmenopausal women without or with VFX on standardized lateral spinal radiographs. Bone biopsy specimens were embedded in methylmethacrylate (MMA). Histomorphometry was done in 8 μm (U.S.A.) or 5 μm (Europe) Goldner stained sections. Vertebral fracture status (yes/no) was the outcome variable in logistic regression models adjusted for age and biopsy specimen origin (U.S.A. vs. Europe). Patients with and without VFX (26/62) were similar regarding age (69.2 ± 5.2 years vs. 67.3 ± 6.7 years), bone volume (BV/TV; 17.7 ± 4.7% vs. 19.0 ± 5.8%), and bone surface (BS/TV; 2.7 ± 0.6 mm2/mm3 vs. 2.8 ± 0.6 mm2/mm3). A lower cortical thickness (C.Th; 652 ± 267 μm vs. 822 ± 325 μm), total strut length (TSL; 826 ± 226 μm/mm2 vs. 922 ± 256 μm/mm2), node-to-loop (Nd-Lp) strut length (10.1 ± 10.3% vs. 15.0 ± 13.6%), together with a higher node-to-terminus (Nd-Tm) strut length (45.6 ± 9.7% vs. 39.1 ± 9.3%) were each associated with prevalent VFX (0.01 < p < 0.10). Differences in BV/TV did not explain these associations. In conclusion, cortical thinning and disruption of trabecular lattice are possible pathogenic mechanisms in patients with VFX.
Hormonal and Biochemical Parameters and Osteoporotic Fractures in Elderly Men
Authors: Dr. Center Jacqueline R. , Nguyen Tuan V. , Sambrook Philip N. , Eisman John A.
Published: April 2006

Low testosterone has been associated with hip fracture in men in some studies. However, data on other hormonal parameters and fracture outcome in men is minimal. This study examined the association between free testosterone (free T) estradiol (E2), sex hormone-binding globulin (SHBG), 25-hydroxyvitamin D [25(OH)D], parathyroid hormone (PTH), insulin-like growth factor I (IGF-I), and fracture in 437 elderly community-dwelling men. Age, height, weight, quadriceps strength, femoral neck bone mineral density (FN BMD), and fracture data (1989–1997) also were obtained. Fractures were classified as major (hip, pelvis, proximal tibia, multiple rib, vertebral, and proximal humerus) or minor (remaining distal upper and lower limb fractures). Fifty-four subjects had a fracture (24 major and 30 minor). There was no association between minor fractures and any hormonal parameter. Risk of major fracture was increased 2-fold for each SD increase in age, decrease in weight and height, and increase in SHBG, and risk of major fracture was increased 3-fold for each SD decrease in quadriceps strength, FN BMD, and 25(OH)D (univariate logistic regression). Independent predictors of major fracture were FN BMD, 2.7 (1.5–4.7; odds ratio [OR]) and 95% confidence interval [CI]); 25(OH)D, 2.8 (1.5–5.3); and SHBG, 1.7 (1.2–2.4). An abnormal value for three factors resulted in a 30-fold increase in risk but only affected 2% of the population. It is not immediately apparent how 25(OH)D and SHBG, largely independently of BMD, may contribute to fracture risk. They may be markers for biological age or health status not measured by methods that are more traditional and as such may be useful in identifying those at high risk of fracture.
Posterior-Anterior and Lateral Dual-Energy X-Ray Absorptiometry for the Assessment of Vertebral Osteoporosis and Bone Loss Among Older Men
Authors: Zmuda Joseph M. , Cauley Jane A. , Glynn Nancy W. , Finkelstein Joel S.
Published: April 2006

Lateral spine dual-energy x-ray absorptiometry (DXA) selectively measures the trabecular-rich vertebral bodies without the contributions of the cortical-rich posterior elements of the spine and is less affected by spinal degenerative disease than posterior-anterior DXA. We tested whether lateral DXA detects vertebral osteoporosis more often and is more sensitive to age-related bone loss than posterior-anterior DXA in 193 healthy, community-dwelling men aged 51–81 years (mean ± SD; 67 ± 8 years). All men had supine lateral, posterior-anterior, and proximal femur DXA scans on a Hologic QDR 2000 densitometer. A subset (n = 102) had repeat scans after 4 years to determine annualized rates of change in bone mineral density (BMD). Age was inversely and significantly associated with BMD in the midlateral (r = −0.27) and lateral (r = −0.24) but not posterior-anterior (r = 0.04) projections. Midlateral (−1.43 ± 3.48% per year; p = 0.0001), lateral (−0.27 ± 1.68% per year; p = 0.12), and hip (−0.19 ± 1.02% per year; p = 0.06) BMD decreased, whereas posterior-anterior BMD increased (0.73 ± 1.11% per year; p = 0.0001) during follow-up. When compared with normal values in 43 men aged 21–42 years, mean T scores were significantly lower with lateral (−1.47 ± 1.32) and midlateral (−1.57 ± 1.36) than posterior-anterior (−0.12 ± 1.30; p < 0.0001) DXA. Only 2.6% of the older men were considered osteoporotic (T score ≤ −2.5) at the posterior-anterior spine, whereas 11.0% were osteoporotic at the femoral neck, 22.5% at the lateral spine, and 24.6% were osteoporotic at the midlateral spine. We conclude that supine lateral DXA identifies considerably more men as osteoporotic and is more sensitive to age-related bone loss than posterior-anterior DXA. Spinal osteoporosis may represent a substantially greater health problem among older men than previously recognized.
ADAM8: A Novel Osteoclast Stimulating Factor
Authors: Choi Sun Jin , Han Je-Ho , Roodman G. David
Published: April 2006

We used polymerase chain reaction (PCR)-selective complementary DNA (cDNA) subtraction hybridization with an immortalized murine osteoclast (OCL) precursor cell line to identify genes that are highly expressed in OCLs compared with OCL precursors and which may be involved in the OCL differentiation process. ADAM8 was one of the 50 genes identified. ADAM (a disintegrin and metalloproteinase) peptides are membrane-bound proteins that can act as cell-to-cell and cell-to-matrix adhesion molecules, degrade the extracellular matrix, and play a role in tissue morphogenesis. Addition of antisense (AS) S-oligonucleotides for ADAM8 (1-10 nM) to mouse bone marrow cultures treated with 10−9 M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] significantly inhibited OCL formation compared with treatment with the control S-oligonucleotide. Furthermore, conditioned media from 293 cells transiently transfected with a secretable form of the ADAM8 cDNA increased OCL formation in a dose-dependent manner. In addition, treatment of OCLs with soluble ADAM8 conditioned media significantly increased pit formation per dentin slice compared with control OCLs. Time course studies indicated that ADAM8 increased OCL formation only when it was present during days 4-7 of the 7-day culture period. Structural analysis, using truncated constructs of ADAM8, showed that the cysteine-rich/disintegrin domain was responsible for its OCL stimulatory activity. Western blot analysis confirmed that the soluble form of ADAM8 is present in normal marrow cultures. These data suggest that ADAM8 plays an important role in OCL formation and acts primarily at the later stages of OCL differentiation.
Role of N-Cadherin and Protein Kinase C in Osteoblast Gene Activation Induced by the S252W Fibroblast Growth Factor Receptor 2 Mutation in Apert Craniosynostosis
Authors: Lemonnier Jérome , Haÿ Eric , Delannoy Philippe , Lomri Abderrahim , Modrowski Dominique , Caverzasio Joseph , Marie Pierre J.
Published: April 2006

Apert (Ap) syndrome is characterized by premature cranial suture ossification caused by fibroblast growth factor receptor 2 (FGFR-2) mutations. We studied the role of cadherins and signaling events in the phenotypic alterations induced by the Ap FGFR-2 S252W mutation in mutant immortalized fetal human calvaria osteoblasts. The FGFR-2 mutation caused increased expression of the osteoblast markers alkaline phosphatase (ALP), type 1 collagen (COLIA1), and osteocalcin (OC) in long-term culture. The mutation also increased cell-cell aggregation, which was suppressed by specific neutralizing anti-N- and anti-E-cadherin antibodies. Mutant osteoblasts showed increased N- and E-cadherin, but not N-cell adhesion molecule (N-CAM) messenger RNA (mRNA) and protein levels. This was confirmed in vivo by the abundant immunoreactive N- and E-cadherins in preosteoblasts in the Ap suture whereas N-CAM and α- and β-catenins were unaffected. Neutralizing anti-N-cadherin antibody or N-cadherin antisense (AS) oligonucleotides but not anti-E-cadherin antibody or AS reduced ALP activity as well as ALP, COLIA1, and OC mRNA overexpression in mutant osteoblasts. Analysis of signal transduction revealed increased phospholipase Cγ (PLCγ) and protein kinase Cα (PKCα) phosphorylation and increased PKC activity in mutant cells in basal conditions. Inhibition of PKC by calphostin C or the PKCα-specific inhibitor Gö6976 suppressed the increased N-cadherin mRNA and protein levels as well as the overexpression of ALP, COLIA1, and OC mRNA in mutant cells. Thus, N-cadherin plays a role in the activation of osteoblast differentiation marker genes in mutant osteoblasts and PKCα signaling appears to be involved in the increased N-cadherin and osteoblast gene expression induced by the S252W FGFR-2 mutation in human osteoblasts.
Variations in Site and Levels of Expression of Chondrocyte Nucleotide Pyrophosphohydrolase with Aging
Authors: Masuda Ikuko , Iyama Ken-Ichi , Halligan Brian D. , Barbieri Joseph T. , Haas Arthur L. , Mccarty Daniel J. , Ryan Lawrence M.
Published: April 2006

The aim of this study was to identify changes in cartilage intermediate layer protein/nucleotide pyrophosphohydrolase (CILP/NTPPH) expression in articular cartilage during aging. Adult (3-4 years old) and young (7-10 days old) porcine articular hyaline cartilage and fibrocartilage were studied by Northern blot analysis, in situ hybridization, and immunohistochemistry using a complementary DNA (cDNA) probe encoding porcine CILP/NTPPH and antibody to a synthetic peptide corresponding to a CILP/NTPPH sequence. Northern blot analysis of chondrocytes showed lower expression of CILP/NTPPH messenger RNA (mRNA) in young cartilage than in adult cartilage. In adult cartilage, extracellular matrix from the surface to the middeep zone was immunoreactive for CILP/NTPPH, especially in the pericellular matrix surrounding the middeep zone chondrocytes. In young cartilage, chondrocytes were moderately immunoreactive for CILP/NTPPH throughout all zones except the calcified zone. The matrix of young cartilage was negative except in the superficial zone. In young cartilage, CILP/NTPPH mRNA expression was undetectable. In adult cartilage, chondrocytes showed strong mRNA expression for CILP/NTPPH throughout middeep zones. Protein and mRNA signals were not detectable below the tidemark. CILP/NTPPH secretion into matrix around chondrocytes increases with aging. In this extracellular site it may generate inorganic pyrophosphate and contribute to age-related calcium pyrophosphate dihydrate crystal deposition disease.
Colocalization of Noggin and Bone Morphogenetic Protein-4 During Fracture Healing
Authors: Yoshimura Yasuo , Nomura Shintaro , Kawasaki Satoshi , Tsutsumimoto Takahiro , Shimizu Tominaga , Takaoka Kunio
Published: April 2006

The regulation of callus formation during fracture repair involves the coordinate expression of growth factors and their receptors. This article describes the temporal and spatial expression of noggin gene, an antagonist to bone morphogenetic protein (BMP), during the fracture repair process. Noggin expression was examined by means of Northern blotting and in situ hybridization and compared with the expression pattern of BMP-4 in a model of fracture repair in adult mice. Expression levels of noggin messenger RNA (mRNA) were enhanced in the early phase of fracture callus formation. The localization of the noggin mRNA was similar to that of BMP-4 mRNA. Distinct noggin mRNA signals were located predominantly in cells lining the periosteum and the cortical endosteum near the fracture site at 2 days after fracture. At 5, 10, and 21 days after fracture, noggin mRNA was detected in the chondrocytes and osteoblasts in the newly formed callus. The pattern of localization was indistinguishable from that of BMP-4. These results suggest that the noggin/BMP-4 balance could be an important factor in the regulation of callus formation during fracture healing.
Classification of Osteoporosis Based on Bone Mineral Densities
Authors: Lu Ying , Genant Harry K. , Shepherd John , Zhao Shoujun , Mathur Ashwini , Fuerst Thomas P. , Cummings Steven R.
Published: April 2006

In this article we examine the role of bone mineral density (BMD) in the diagnosis of osteoporosis. Using information from 7671 women in the Study of Osteoporotic Fractures (SOF) with BMD measurements at the proximal femur, lumbar spine, forearm, and calcaneus, we examine three models with differing criteria for the diagnosis of osteoporosis. Model 1 is based on the World Health Organization (WHO) criteria using a T score of −2.5 relative to the manufacturers' young normative data aged 20-29 years, with modifications using information from the Third National Health and Nutrition Examination Survey (NHANES). Model 2 uses a T score of −1 relative to women aged 65 years at the baseline of the SOF population. Model 3 classifies women as osteoporotic if their estimated osteoporotic fracture risk (spine and/or hip) based on age and BMD is above 14.6%. We compare the agreement in osteoporosis classification according to the different BMD measurements for the three models. We also consider whether reporting additional BMD parameters at the femur or forearm improves risk assessment for osteoporotic fractures. We observe that using the WHO criteria with the manufacturers' normative data results in very inconsistent diagnoses. Only 25% of subjects are consistently diagnosed by all of the eight BMD variables. Such inconsistency is reduced by using a common elderly normative population as in model 2, in which case 50% of the subjects are consistently diagnosed as osteoporotic by all of the eight diagnostic methods. Risk-based diagnostic criteria as in model 3 improve consistency substantially to 68%. Combining the results of BMD assessments at more than one region of interest (ROI) from a single scan significantly increases prediction of hip and/or spine fracture risk and elevates the relative risk with increasing number of low BMD subregions. We conclude that standardization of normative data, perhaps referenced to an older population, may be necessary when applying T scores as diagnostic criteria in patient management. A risk-based osteoporosis classification does not depend on the manufacturers' reference data and may be more consistent and efficient for patient diagnosis.
Functional Impact of Human Collagen α2(XI) Gene Polymorphism in Pathogenesis of Ossification of the Posterior Longitudinal Ligament of the Spine
Authors: Maeda Shingo , Ishidou Yasuhiro , Koga Hiroaki , Taketomi Eiji , Ikari Katsunori , Komiya Setsuro , Takeda Jun , Sakou Takashi , Inoue Ituro
Published: April 2006

Ossification of the posterior longitudinal ligament (OPLL) of the spine is the leading cause of myelopathy in Japan. In earlier studies, we provided genetic linkage and allelic association evidence of distinct differences in the human collagen α2(XI) gene (COL11A2) that might constitute inherited predisposition to OPLL.(1) In the present study, a strong allelic association with non-OPLL (p = 0.0003) was observed with an intron 6 polymorphism [intron 6 (−4A)], in which the intron 6 (−4A) allele is more frequently observed in non-OPLL subjects than in OPLL patients. In addition, a newly identified polymorphism in exon 6 [exon 6 (+28A)] was in linkage disequilibrium with the intron 6 (−4A). The functional impact of the polymorphisms was analyzed by comparing the differences in messenger RNA (mRNA) splicing by reverse-transcription polymerase chain reaction (RT-PCR) analysis in cultured cells from the interspinous ligament and an in vitro exon trapping study. The intron 6 (−4A) allele resulted in skipping exon 6 and retaining exon 7, while the exon 6 (+28A) allele was not associated with alteration in mRNA splicing. Similar mRNA species were observed in undifferentiated osteoblast (Ob) cells and in cells from posterior longitudinal ligament of non-OPLL subjects. The region containing exons 6-8 is an acidic subdomain presumably exposed to the surface that could interact with molecules of the extracellular matrix. Accordingly, retaining exon 7 together with removal of exon 6 observed in intron 6 (−4A) could play a protective role in the ectopic ossification process because the same pattern was observed in undifferentiated Ob cells and nonossified posterior longitudinal ligament cells.
Establishment of an Osteoid Preosteocyte-like Cell MLO-A5 That Spontaneously Mineralizes in Culture
Authors: Kato Y. , Boskey A. , Spevak L. , Dallas M. , Hori M. , Bonewald L. F.
Published: April 2006

The mechanisms controlling the initiation of mineralization of bone matrix are not clear. To examine this process, we established a cell line called MLO-A5 that mineralizes in sheets, not nodules, within 3 days of culture in the presence of β-glycerophosphate (β-GP) and ascorbic acid and within 7 days in the absence of β-GP and ascorbic acid. The mineral formed in both cases was shown to be bonelike apatite by Fourier transformed infrared (FTIR) spectroscopy. Mineral-to-matrix ratios (min/matrix) calculated from the FTIR data, which are related directly to ash weight, were approximately 0.4 in the absence of β-GP and ascorbic acid and approximately 1.2 in the presence of β-GP and ascorbic acid. By comparison, these ratios in fetal rat calvarial cells without β-GP equal 0 and with β-GP 1.9. This cell line and three others (MLO-A2, −D1, and −D6) were isolated from the long bones of transgenic mice expressing the large T-antigen driven by the osteocalcin promoter, the same mice from which the osteocyte-like cell line MLO-Y4 was isolated.(1) The cell lines were selected based on a dendritic or stellate morphology. MLO-A5 cells express high alkaline phosphatase, collagen type 1, parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor, bone sialoprotein (BSP), and osteocalcin (767 ng/106 cells compared with <1–2.2 ng/106 cell for primary mouse osteoblasts and five osteoblast cell lines). The single unique feature of the MLO-A5 cells compared with the other three nonmineralizing cell lines is the high expression of messenger RNA (mRNA) for BSP. These cell lines may represent stages of osteocyte differentiation and the MLO-A5 cells represent the postosteoblast, preosteocyte responsible for triggering mineralization of osteoid.
Human Parathyroid Hormone 1–34 Reverses Bone Loss in Ovariectomized Mice
Authors: Alexander J. M. , Bab I. , Fish S. , Müller R. , Uchiyama T. , Gronowicz G. , Nahounou M. , Zhao Q. , White D. W. , Chorev M. , Gazit D. , Rosenblatt M.
Published: April 2006

The experimental work characterizing the anabolic effect of parathyroid hormone (PTH) in bone has been performed in nonmurine ovariectomized (OVX) animals, mainly rats. A major drawback of these animal models is their inaccessibility to genetic manipulations such as gene knockout and overexpression. Therefore, this study on PTH anabolic activity was carried out in OVX mice that can be manipulated genetically in future studies. Adult Swiss-Webster mice were OVX, and after the fifth postoperative week were treated intermittently with human PTH(1–34) [hPTH(1–34)] or vehicle for 4 weeks. Femoral bones were evaluated by microcomputed tomography (μCT) followed by histomorphometry. A tight correlation was observed between trabecular density (BV/TV) determinations made by both methods. The BV/TV showed >60% loss in the distal metaphysis in 5-week and 9-week post-OVX, non-PTH-treated animals. PTH induced a ∼35% recovery of this loss and a ∼40% reversal of the associated decreases in trabecular number (Tb.N) and connectivity. PTH also caused a shift from single to double calcein-labeled trabecular surfaces, a significant enhancement in the mineralizing perimeter and a respective 2- and 3-fold stimulation of the mineral appositional rate (MAR) and bone formation rate (BFR). Diaphyseal endosteal cortical MAR and thickness also were increased with a high correlation between these parameters. These data show that OVX osteoporotic mice respond to PTH by increased osteoblast activity and the consequent restoration of trabecular network. The Swiss-Webster mouse model will be useful in future studies investigating molecular mechanisms involved in the pathogenesis and treatment of osteoporosis, including the mechanisms of action of known and future bone antiresorptive and anabolic agents.
RGD Peptides Immobilized on a Mechanically Deformable Surface Promote Osteoblast Differentiation
Authors: Cavalcanti-Adam E. A. , Shapiro I. M. , Composto R. J. , Macarak E. J. , Adams C. S.
Published: April 2006

The major objective of this work was to attach bone cells to a deformable surface for the effective transmission of force. We functionalized a silastic membrane and treated it with 3-aminopropyltriethoxysilane (APTS). A minimal RGD peptide was then covalently linked to the aminated surface. MC3T3-E1 osteoblast-like cells were cultured on the arginine-glycine-aspartic acid (RGD)-treated membrane for 3–15 days and cell attachment and proliferation was evaluated. We observed that cells were immediately bound to the membrane and proliferated. After 8 days on the material surface, osteoblasts exhibited high levels of ALP staining, indicating that the cells were undergoing maturation. Alizarin red staining and Fourier transform infrared (FTIR) analysis showed that the mineral formed by the cells was a biological apatite. The second objective was to apply a mechanical force to cells cultured on the modified silicone membrane. Dynamic equibiaxial strain, 2% magnitude, and a 0.25-Hz frequency were applied to bone cells for 2 h. Osteoblasts elicited increased phalloidin fluorescence, suggesting that there was reorganization of the cytoskeleton. Furthermore, the applied strain elicited increased expression of the αvβ3 integrin receptor. We concluded that the covalent binding of RGD peptides to a silicone membrane provides a compatible surface for the attachment and subsequent differentiation of osteoblasts. Moreover, the engineered surface transduces applied mechanical forces directly to the adherent cells via integrin receptors.
Bone Morphogenetic Protein-Mediating Receptor-Associated Smads as well as Common Smad Are Expressed in Human Articular Chondrocytes but not Up-Regulated or Down-Regulated in Osteoarthritic Cartilage
Authors: Bau Brigitte , Haag Jochen , Schmid Erik , Kaiser Martina , Gebhard Pia Margarethe , Aigner Thomas
Published: April 2006

Bone morphogenetic proteins (BMPs) are supposed to be important for cartilage matrix anabolism. In this study, we investigated whether the intracellular mediators of BMP activity, Smads 1, 4, 5, and 8, are expressed in normal human articular chondrocytes in vivo and in vitro and whether alterations in expression and distribution pattern are found in osteoarthritic cartilage or in vitro after stimulation with interleukin (IL)-1, because down-regulation of these mediators could be responsible for the decrease of anabolic activity in osteoarthritic cartilage. RNA was isolated from normal and osteoarthritic human knee cartilage and analyzed by (quantitative) polymerase chain reaction (PCR) technology. Articular chondrocytes were cultured in alginate beads and short-term high-density monolayer cultures with and without stimulation by IL-1. In addition, immunolocalization of the receptor-associated Smads (R-Smads) was performed on sections of normal and diseased articular cartilage. Reverse-transcription (RT)-PCR analysis showed a moderate expression of all Smads investigated in normal, early degenerative, and late stage osteoarthritic cartilage. Immunolocalization detected the R-Smads in most chondrocytes on the protein level in all specimen groups investigated. In vitro, the Smads were also expressed and partly up-regulated by Il-1β in alginate bead culture. Of note, for Smad 1, two truncated splice variants were expressed by articular chondrocytes missing exon 4 as well as exons 3 and 4. Our study showed that BMP-receptor Smads 1, 5, and 8 as well as common Smad (C-Smad) 4 are expressed and present in human normal and osteoarthritic articular chondrocytes corroborating the importance of BMPs and BMP signaling for articular cartilage. This study is the first to describe splicing variants for Smad 1. Smads 1, 4, and 5 are up-regulated in vitro by Il-1β, suggesting a linkage of the Il-1 and BMP-signaling pathways within the chondrocytes. None of the Smads were grossly up- or down-regulated in osteoarthritic chondrocytes, suggesting that differences in overall expression levels of the investigated Smad proteins are not relevant for metabolic activity of articular chondrocytes in vivo.
The Relationship Between Bone Density and Incident Vertebral Fracture in Men and Women
Authors:   The European Prospective Osteoporosis Study (EPOS) Group, O'Neill TW
Published: April 2006

Bone mineral density (BMD) is an important predictor of future fracture risk in women; however, there are few prospective data in men. The aim of this analysis was to determine whether there are differences in the relationship between BMD and incident vertebral fracture in men and women. Men and women were recruited from population-based registers in 21 European centers. Those recruited were interviewed and had spinal radiographs performed. The radiographs were assessed morphometrically and prevalent vertebral deformity was defined using the McCloskey-Kanis method. Repeat spinal radiographs were performed at a mean of 3.8 years after the baseline radiographs. Incident fractures were defined using a combination of the point prevalence and 20% reduction in vertebral height (plus a 4-mm reduction in absolute height) criteria. BMD measurements were made in a subsample of those recruited. Poisson regression was used to explore the influence of gender, age, prevalent deformity, and BMD on the incidence of vertebral fracture. Thirty-four hundred sixty-one men and women had both paired spinal radiographs and bone density measurements performed. BMD at the spine and femoral neck was higher in men than in women. After adjusting for age, the risk of incident vertebral fracture was greater in women than in men (relative risk [RR] = 2.3; 95% CI, 1.5–3.6) and increased by a factor of 1.4 (95% CI, 1.2–1.8), 1.5 (95% CI, 1.2–1.8), and 1.6 (95% CI, 1.3–1.9) per decrease of 0.1 g/cm2 in BMD at the spine, femoral neck, and trochanter, respectively. After adjusting for BMD at the spine or trochanter, the gender difference in the predicted age-specific incidence of vertebral fracture was no longer significant (RR = 1.1 and 95% CI, 0.6–1.9 at the spine; RR = 1.5 and 95% CI, 0.8–2.7 at the trochanter), although it persisted after adjusting for femoral neck BMD (RR = 1.9; 95%CI, 1.1–3.3). The presence of a prevalent vertebral deformity was a strong risk factor for future vertebral fracture, although the strength of the association was reduced after adjustment for age, sex, and spine BMD. However, adjustment for the presence of a baseline vertebral deformity did not alter the main findings. In conclusion, at a given age and spine (although not femoral neck) bone density, the risk of incident vertebral fracture is similar in men and women. Incident vertebral fractures are more common in women than men because at any age their spine BMD is lower.
Osteoporosis in Men and Women: A Story About Bone Mineral Density Thresholds and Hip Fracture Risk
Authors: de Laet Chris E. D. H. , Van Der Klift Marjolein , Hofman Albert , Pols Huibert A. P.
Published: April 2006

In postmenopausal women, the T score for bone mineral density (BMD) is a well-accepted diagnostic criterion for osteoporosis. It is also used to assess fracture risk. However, it is unclear whether in elderly men similar BMD thresholds should be used. Different hypotheses have been proposed for the relation of BMD with hip fracture risk in men. In this study, we tested those hypotheses using a mathematical model and we compared the calculated results with observed prospective data from the Rotterdam study. In the model, we combined the observed femoral neck BMD distribution for men and women with previously derived hip fracture risk functions based on age and BMD. For men, we tested different hypotheses for the relation of BMD with hip fracture risk. The relation of BMD with hip fracture risk is similar in men and women (scenario 1) or the relative risk (RR) per standard deviation (SD) decrease of BMD is either larger or smaller in men than in women (scenario 2a and 2b), or, at a similar absolute fracture risk, men have a higher BMD (scenario 3). In the prospective data, men with a hip fracture had an average BMD that was 0.070 g/cm2 higher than women with a hip fracture. The calculated results from the first scenario were consistent with those data and were also consistent with the observed hip fracture incidence and the observed female-to-male (F/M) risk ratio (1.7). When the RR for each SD decrease of BMD was assumed to be either larger or smaller in men than in women (second scenario), the calculated average BMD difference in men and women became respectively smaller or larger than observed. When men would have a higher fracture risk at similar BMD levels (third scenario), the calculated total number of hip fractures increased and even exceeded that in women, with an F/M risk ratio of 0.94 in our example. In women, a larger proportion of hip fractures occurs at a T score below −2.5 than in men using the same absolute BMD threshold, but using a male-specific T score largely solves this diagnostic problem. Taken together, the average hip fracture risk in men is much lower than in women but appeared to be similar at the same BMD. Therefore, we propose the use of the same absolute BMD thresholds for decisions about interventions.
Long-Term Dosing of Arzoxifene Lowers Cholesterol, Reduces Bone Turnover, and Preserves Bone Quality in Ovariectomized Rats
Authors: Ma Yanfei L. , Bryant Henry U. , Zeng Qingqiang , Palkowitz Alan , Jee Webster S. S. , Turner Charles H. , Sato Masahiko
Published: April 2006

Long-term effects of a new selective estrogen receptor modulator (SERM) arzoxifene were examined in ovariectomized (OVX) rats. Arzoxifene was administered postoperatively (po) at 0.1 mg/kg per day or 0.5 mg/kg per day to 4-month-old rats, starting 1 week after OVX for 12 months. At study termination, body weights for arzoxifene groups were 16–17% lower than OVX control, which was caused by mainly reduced gain of fat mass. Longitudinal analysis of the proximal tibial metaphysis (PTM) by computed tomography (CT) at 0, 2, 4, 6, 9, and 12 months showed that OVX induced a 22% reduction in bone mineral density (BMD) at 2 months, which narrowed to a 12% difference between sham-operated (sham) and OVX rats by 12 months. Both doses of arzoxifene prevented the OVX-induced decline in BMD. Histomorphometry of the PTM showed that arzoxifene prevented bone loss by reducing osteoclast number in OVX rats. Arzoxifene maintained bone formation indices at sham levels and preserved trabecular number above OVX controls. Micro-CT analysis of lumbar vertebrae showed similar preservation of BMD compared with OVX, which were not different from sham. Compression testing of the vertebra and three-point bending testing of femoral shaft showed that strength and toughness were higher for arzoxifene-treated animals compared with OVX animals. Arzoxifene reduced serum cholesterol by 44–59% compared with OVX. Uteri wet weight from arzoxifene animals was 38–40% of sham compared with OVX rats, which were 29% of sham. Histology of the uterine endometrium showed that cell heights from both doses of arzoxifene were not significantly different from OVX controls. In summary, treatment of OVX rats with arzoxifene for nearly one-half of a lifetime maintained beneficial effects on cholesterol and the skeleton. These data suggest that arzoxifene may be a useful therapeutic agent for osteoporosis in postmenopausal women.
c-myc Is Required for Osteoclast Differentiation
Authors: Battaglino R. , Kim D. , Fu J. , Vaage B. , Fu X-Y. , Stashenko P.
Published: April 2006

The role of the receptor activator of nuclear factor κB (NF-κB) ligand (RANKL)—a tumor necrosis factor (TNF)-related cytokine—in osteoclast formation has been established clearly. However, the downstream signaling pathways activated by this cytokine remain largely unknown. To identify genes that play a role in osteoclastogenesis, we used RAW 264.7 mouse monocytes as a model system for the differentiation of multinucleated osteoclasts from mononucleated precursors. RAW 264.7 cells were induced with RANKL to form multinucleated giant osteoclast-like cells (OCLs) that expressed a number of osteoclast-specific markers and were able to form resorption pits on both calcium phosphate films and bone slices. This system was used to identify genes that are regulated by RANKL and may play a role in osteoclast differentiation. The proto-oncogene c-myc was strongly up-regulated in RANKL-induced OCLs but was absent in undifferentiated cells. Expression of Myc partners Max and Mad, on the other hand, was constant during OCL differentiation. We expressed a dominant negative Myc in RAW 264.7 cells and were able to block RANKL-induced OCL formation. Northern Blot analysis revealed a delay and a significant reduction in the level of messenger RNA (mRNA) for tartrate-resistant acid phosphatase (TRAP) and cathepsin K. We conclude that c-myc is a downstream target of RANKL and its expression is required for RANKL-induced osteoclastogenesis.
Involvement of Cyclo-Oxygenase-2 in Osteoclast Formation and Bone Destruction in Bone Metastasis of Mammary Carcinoma Cell Lines
Authors: Ono Katsuhiro , Akatsu Takuhiko , Murakami Takehiko , Kitamura Ryuichi , Yamamoto Michiko , Shinomiya Nariyoshi , Rokutanda Makoto , Sasaki Tomoyo , Amizuka Norio , Ozawa Hidehiro , Nagata Naokazu , Kugai Nobuo
Published: April 2006

We previously reported that mouse mammary carcinoma cell lines (MMT060562 and BALB/c-MC) induced osteoclast formation through production of prostaglandin E2 (PGE2) in cocultures with mouse bone marrow cells, but the mechanism(s) of PG production remained unclear. In the present in vitro and in vivo studies, we tested the involvement of cyclo-oxygenase-2 (COX-2), an inducible rate-limiting enzyme in PG biosynthesis, in the stimulation of osteoclast formation by mouse mammary carcinoma cell lines. Addition of a selective COX-2 inhibitor, JTE-522, to cocultures of mammary carcinoma cell lines and bone marrow cells lowered PGE2 concentration in the culture media and inhibited osteoclast formation in a dose-dependent manner. Northern blotting showed a very high level of COX-2 messenger RNA (mRNA) expression in MMT060562. The mRNA expression was low in BALB/c-MC, but it increased when BALB/c-MC and bone marrow cells were cocultured. The results of immunocytochemistry for COX-2 protein in respective cultures were compatible with the results of COX-2 mRNA. In vivo, BALB/c-MC injected into the heart of Balb/c mice metastasized to bone and formed osteolytic lesions in their hindlimbs. Histological examination revealed that tumor cells had metastasized to the bone marrow cavity and destroyed the bone trabeculae. Immunohistochemistry demonstrated that bone marrow stromal cells adjacent to tumor cells expressed COX-2 protein. These findings suggest that COX-2 plays an important role in the osteolysis of bone metastasis in vivo as well as in osteoclast formation in cocultures used as an in vitro model of metastatic bone disease.
Prostaglandin E2 Induces Interaction Between hSlo Potassium Channel and Syk Tyrosine Kinase in Osteosarcoma Cells
Authors: Rezzonico Roger , Schmid-Alliana Annie , Romey Georges , Bourget-Ponzio Isabelle , Breuil Véronique , Breittmayer Violette , Tartare-Deckert Sophie , Rossi Bernard , Schmid-Antomarchi Heidy
Published: April 2006

Prostaglandins (PGs) are important mediators of bone response to growth factors, hormones, inflammation, or mechanical strains. In this study, we show that in MG63 osteosarcoma cells, prostaglandin E2 (PGE2) produces the opening of a large conductance Ca2+-dependent K+ channel (BK). This PGE2-mediated channel opening induces the recruitment of various tyrosine-phosphorylated proteins on the hSlo α-subunit of BK. Because the C-terminal domain of hSlo encompasses an immunoreceptor tyrosine-based activation motif (ITAM), we show that the Syk nonreceptor tyrosine kinase, reported yet to be expressed mainly in hematopoietic cells, is expressed also in osteoblastic cells, and recruited on this ITAM after a PGE2-induced docking/activation process. We show that Syk/hSlo association is dependent of an upstream Src-related tyrosine kinase activity, in accord with the classical two-step model described for immune receptors. Finally, we provide evidence that this Syk/hSlo interaction does not affect the electrical features of BK channels in osteosarcoma cells. With these data, we would like to suggest the new notion that besides its conductance function, hSlo channel can behave in bone cells, as a true transduction protein intervening in the bone remodeling induced by PGE2.
Spatial Distribution of Bax and Bcl-2 in Osteocytes After Bone Fatigue: Complementary Roles in Bone Remodeling Regulation?
Authors: Verborgt Olivier , Tatton Nadine A. , Majeska Robert J. , Schaffler Mitchell B.
Published: April 2006

Osteocyte apoptosis appears to play a key role in the mechanism by which osteoclastic resorption activity targets bone for removal, because osteocyte apoptosis occurs in highly specific association with microdamage and subsequent remodeling after fatigue. However, beyond terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) assay, little is known about the mechanisms controlling osteocyte apoptosis in vivo. In the current studies, expression of Bax, a proapoptotic gene product, and Bcl-2, an antiapoptotic gene product, was determined in osteocytes of fatigued rat bone using immunocytochemical staining and compared with TUNEL staining patterns. Bax and Bcl-2 were evident in osteocytes by 6 h after loading. Moreover, Bax and Bcl-2 in osteocytes were expressed differently as a function of distance from microdamage sites. The peak of Bax expression and TUNEL+ staining in osteocytes was observed immediately at the microcrack locus, which is where bone resorption occurs in this system; in contrast, Bcl-2 expression, the antiapoptotic signal, reached its greatest level at some distance (1-2 mm) from microcracks. These data suggest that near sites of microinjury in bone, those osteocytes that do not undergo apoptosis are prevented from doing so by active protection mechanisms. Moreover, the zone of apoptotic osteocytes around microcracks was effectively “walled in” by a surrounding halo of surviving osteocytes actively expressing Bcl-2. Thus, the expression pattern of apoptosis-inhibiting gene products by osteocytes surrounding the apoptotic osteocyte at microdamage sites also may provide important signals in the guidance of resorption processes that occur in association with osteocyte apoptosis after fatigue.
The Incidence of Vertebral Fractures in Men and Women: The Rotterdam Study
Authors: Van Der Klift Marjolein , De Laet Chris E. D. H. , Mccloskey Eugene V. , Hofman Albert , Pols Huibert A. P.
Published: April 2006

Vertebral fractures are considered the most common fractures in osteoporosis. Nevertheless, little is known about the epidemiology of these fractures, especially in men. Therefore, the incidence of vertebral fractures was studied in 3469 men and women from the Rotterdam Study. Spinal radiographs were obtained at baseline and again after a mean follow-up of 6.3 years. The follow-up radiographs were scored for vertebral fractures using the McCloskey-Kanis assessment method. Whenever a vertebral fracture was detected, the radiograph was compared with the baseline radiograph. If this fracture was not already present at baseline, it was considered an incident fracture. The incidence increased strongly with age, ranging from 7.8/1000 person years (PY) at ages 55-65 years to 19.6/1000 PY at ages over 75 years for women, and 5.2-9.3/1000 PY for men, respectively. Analyses repeated in strata of presence or absence of prevalent vertebral fractures showed that both in men and in women, the increase in incidence with age was almost exclusively observed in subjects with one or more prevalent fractures present at baseline. For both genders, the incidence of vertebral fractures doubled per SD decrease in lumbar spine or femoral neck bone mineral density (BMD). This study shows that overall, the incidence of vertebral fractures is higher in women than in men. In both genders, the incidence increases with age. Furthermore, the presence of a prevalent vertebral fracture and a low BMD are strong independent predictors of incident vertebral fractures in men and women.