bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of CCAAT Enhancer Binding Protein Transcription Factors in the Regulation of Prostaglandin G/H Synthase 2 Expression by Interleukin-1 in Osteoblastic MC3T3-E1 Cells
Authors: Harrison John R. , Kelly Penny L. , Pilbeam Carol C.
Published: April 2006

Interleukin-1 (IL-1) stimulates prostaglandin production in bone by a rapid and transient activation of prostaglandin G/H synthase 2 (PGHS-2) gene expression. In osteoblastic MC3T3-E1 cells, IL-1 caused a transient increase in PGHS-2 messenger RNA (mRNA), which peaked at 2 h. IL-1 caused a 2- to 4-fold activation of a 371-base pair (bp) murine PGHS-2 promoter/luciferase construct in stable transfectants. This response mapped to a proximal promoter element(s) located between −150 and −40 bp. This region contains a putative CCAAT enhancer binding protein (C/EBP) site (centered at −135 bp), which shows enhanced binding of C/EBPβ and C/EBPδ by mobility shift analysis after IL-1 treatment. A transient cotransfection approach was used to examine the effects of C/EBPβ and C/EBPδ overexpression. IL-1 caused a maximal 3- to 7-fold stimulation of PGHS-2 promoter activity after 2.5 h. Overexpression of murine C/EBPβ and C/EBPδ caused a dose-dependent increase in basal and IL-1-stimulated luciferase activity. C/EBPδ caused a greater enhancement of basal and IL-1-stimulated promoter activity than C/EBPβ, suggesting that C/EBPδ is a stronger transactivator. Overexpression of p20C/EBPβ, a dominant negative inhibitor of C/EBP function, blocked the stimulation of PGHS-2 promoter activity by IL-1 and blocked the ability of overexpressed C/EBPβ and C/EBPδ to increase basal and IL-1-stimulated promoter activity. Mutagenesis of the C/EBP site reduced, but did not abolish, the stimulation of PGHS-2 promoter activity by IL-1 and blunted the effect of overexpressed C/EBPδ on basal and IL-1-stimulated promoter activity. These results suggest an essential role for C/EBPβ and C/EBPδ in the induction of PGHS-2 gene expression by IL-1 in osteoblastic cells.
Making Rats Rise to Erect Bipedal Stance for Feeding Partially Prevented Orchidectomy-Induced Bone Loss and Added Bone to Intact Rats
Authors: Yao Wei , Jee Webster S. S. , Chen Jianliang , Liu Haoyu , Tam Cherk S. , Cui Liao , Zhou Hua , Setterberg Rebecca B. , Frost Harold M.
Published: April 2006

The objectives of this study were to investigate the different effects on muscle mass and cancellous (proximal tibial metaphysis [PTM]) and cortical (tibial shaft [TX]) bone mass of sham-operated and orchidectomized (ORX) male rats by making rats rise to erect bipedal stance for feeding. Specially designed raised cages (RC) were used so that the rats had to rise to erect bipedal stance to eat and drink for 12 weeks. Dual-energy X-ray absorptiometry (DEXA) and peripheral quantitative computerized tomography (pQCT) were used to estimate the lean leg mass and bone mineral. Static and dynamic histomorphometry were performed on the triple-labeled undecalcified sections. We found that making the intact rats rise to erect bipedal stance for feeding increased muscle mass, cortical bone volume, and periosteal bone formation. Orchidectomy increased net losses of bone next to the marrow by increasing bone turnover. Making the ORX rats rise to erect bipedal stance increased muscle mass, partially prevented cancellous bone loss in the PTM, and prevented net cortical bone loss in TX induced by ORX by depressing cancellous and endocortical high bone turnover and stimulating periosteal bone formation. The bone-anabolic effects were achieved mainly in the first 4 weeks in the PTM and by 8 weeks in the TX. These findings suggested that making the rats rise to erect bipedal stance for feeding helped to increase muscle mass and cortical bone mass in the tibias of intact rats, increase muscle mass, and partially prevented cancellous and net cortical bone loss in ORX rats.
The Effects of Bone Marrow Transplantation on X-linked Hypophosphatemic Mice
Authors: Miyamura Takako , Tanaka Hiroyuki , Inoue Masaru , Ichinose Youjirou , Seino Yoshiki
Published: April 2006

The genes responsible for X-linked hypophosphatemic (XLH) vitamin D-resistant rickets and the murine homolog, hypophosphatemic mice (Hyp), were identified as PHEX and Phex (phosphate-regulating gene with homology to endopeptidases on the X chromosome), respectively. However, the mechanism by which inactivating mutations of PHEX cause XLH remains unknown. We investigated the mechanisms by syngeneic bone marrow transplantation (BMT) from wild mice to Hyp mice. The expression of the Phex gene was detected in mouse BM cells. BMT introduced a chimerism in recipient Hyp mice and a significant increase in the serum phosphorus level. The renal sodium phosphate cotransporter gene expression was significantly increased. The effect of BMT on the serum phosphorus level depended on engraftment efficiencies, which represent the dosage of normal gene. Similarly, the serum alkaline phosphatase (ALP) activity was decreased and bone mineral density was increased. Furthermore, the renal expression of 25-hydroxyvitamin D3 24-hydroxylase, which is a key enzyme in the catabolic pathway and is increased in XLH/Hyp, was improved. From these results, we conclude that transplantation of normal BM cells improved abnormal bone mineral metabolism and deranged vitamin D metabolism in Hyp by replacing defective gene product(s) with normal gene product(s). This result may provide strong evidence for clinical application of BMT in metabolic bone disorders.
Fibroblastic Stromal Cells Express Receptor Activator of NF-κB Ligand and Support Osteoclast Differentiation
Authors: Dr. Quinn Julian M. W. , Horwood Nicole J. , Elliott Jan , Gillespie Matthew T. , Martin T. John
Published: April 2006

Osteoclast formation in bone is supported by osteoblasts expressing receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) expression. Numerous osteotropic factors regulate expression levels of RANKL and the RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts, thereby affecting osteoclast differentiation. However, not only is RANKL widely expressed in soft tissues, but osteoclasts have been noted in extraskeletal lesions. We found that cultured skin fibroblastic cells express RANKL, M-CSF, and OPG messenger (mRNA). Stimulation by 1α,25 dihydroxyvitamin D3 [1,25(OH)2D3] plus dexamethasone (Dex) augmented RANKL and diminished OPG mRNA expression in fibroblastic cells and caused the formation of numerous osteoclasts in cocultures of skin fibroblastic cells with hemopoietic cells or monocytes. The osteoclasts thus formed expressed tartrate-resistant acid phosphatase (TRAP) and calcitonin (CT) receptors and formed resorption pits in cortical bone. Osteoclast formation also was stimulated (in the presence of Dex) by prostaglandin E2 (PGE2), interleukin-11 (IL-11), IL-1, tumor necrosis factor-α (TNF-α), and parathyroid hormone-related protein (PTHrP), factors which also stimulate osteoclast formation supported by osteoblasts. In addition, granulocyte-macrophage-CSF (GM-CSF), transforming growth factor-β (TGF-β), and OPG inhibited osteoclast formation in skin fibroblastic cell-hemopoietic cell cocultures; CT reduced only osteoclast nuclearity. Fibroblastic stromal cells from other tissues (lung, respiratory diaphragm, spleen, and tumor) also supported osteoclast formation. Thus, RANKL-positive fibroblastic cells in extraskeletal tissues can support osteoclastogenesis if osteolytic factors and osteoclast precursors are present. Such mesenchymally derived cells may play a role in pathological osteolysis and may be involved in osteoclast formation in extraskeletal tissues.
Integrin and Mechanosensitive Ion Channel-Dependent Tyrosine Phosphorylation of Focal Adhesion Proteins and β-Catenin in Human Articular Chondrocytes After Mechanical Stimulation
Authors: Lee H. S. , Millward-Sadler S. J. , Wright M. O. , Nuki G. , Dr. Salter D. M.
Published: April 2006

Mechanical forces influence chondrocyte metabolism and function. We have previously shown that 0.33 Hz cyclical pressure-induced strain (PIS) results in membrane hyperpolarization of normal human articular chondrocytes (HAC) by activation of Ca2+-dependent K+ small conductance potassium activated calcium (SK) channels. The mechanotransduction pathway involves α5β1-integrin, stretch-activated ion channels (SAC) actin cytoskeleton and tyrosine protein kinases, with subsequent release of the chondroprotective cytokine interleukin-4 (IL-4). The objective of this study was to examine in detail tyrosine phosphorylation events in the mechanotransduction pathway. The results show tyrosine phosphorylation of three major proteins, p125, p90, and p70 within 1 minute of onset of mechanical stimulation. Immunoblotting and immunoprecipitation show these to be focal adhesion kinase (pp125FAK), β-catenin, and paxillin, respectively. Tyrosine phosphorylation of all three proteins is inhibited by RGD containing oligopeptides and gadolinium, which is known to block SAC. β-catenin coimmunoprecipitates with FAK and is colocalized with α5-integrin and pp125FAK. These results indicate a previously unrecognized role for an integrin-β-catenin signaling pathway in human articular chondrocyte (HAC) responses to mechanical stimulation.
Six-Month Daily Administration of Parathyroid Hormone and Parathyroid Hormone—Related Protein Peptides to Adult Ovariectomized Rats Markedly Enhances Bone Mass and Biomechanical Properties: A Comparison of Human Parathyroid Hormone 1–34, Parathyroid Hormone-Related Protein 1–36, and SDZ-Parathyroid Hormone 893
Authors: Stewart A. F. , Cain R. L. , Burr D. B. , Jacob D. , Turner C. H. , Hock J. M. , Drezner Marc K.
Published: April 2006

Daily administration of parathyroid hormone (PTH) and PTH-related protein (PTHrP) peptides has been shown to increase bone mass and strength in animals and, for PTH, to increase bone mass in humans. Long-term direct comparison of multiple members of the PTH/PTHrP family in vivo has not been reported. We therefore selected three PTH/PTHrP molecules for direct comparison in vivo in an adult rat model of postmenopausal osteoporosis: PTH(1-34), PTHrP(1-36), and the PTH analog, SDZ-PTH 893 {Leu8, Asp10, Lys11, Ala16, Gln18, Thr33, Ala34 human PTH 1–34 [hPTH(1-34)]}. A 6-month study was performed in which adult (6-month-old) vehicle-treated ovariectomized (OVX) and sham OVX rats were compared with OVX rats receiving 40 μg/kg per day of either PTH(1-34), PTHrP(1-36), or PTH-SDZ-893. Bone mass, as assessed by ash weight and densitometry, bone histomorphometry, biomechanical properties at trabecular and cortical sites, and indices of bone formation markedly increased in all three PTH/PTHrP peptide-treated groups as compared with controls. In general, this improvement followed a rank order of SDZ-PTH-893 > PTH > PTHrP. The adverse effect profile also was greatest with SDZ-PTH-893; these rats developed moderate hypercalcemia, marked renal calcium accumulation, and displayed a 13% mortality. These studies show that PTH(1-34), PTHrP(1-36), and PTH-SDZ-893 significantly and progressively increase bone mass and bone strength in this rat model of postmenopausal osteoporosis. The adverse effect profile correlates in general terms with efficacy. All three peptides show promise as skeletal anabolic agents. Further studies in humans will be required to define optimal efficacy-to-adverse effect ratios and relative efficacy for each peptide in human osteoporosis.
Cytokines and Bone Loss in a 5-Year Longitudinal Study—Hormone Replacement Therapy Suppresses Serum Soluble Interleukin-6 Receptor and Increases Interleukin-1-Receptor Antagonist: The Danish Osteoporosis Prevention Study
Authors: Abrahamsen Bo , Bonnevie-Nielsen Vagn , Ebbesen Ebbe Nils , Gram Jeppe , Beck-Nielsen Henning
Published: April 2006

The proinflammatory cytokines interleukin-1β (IL-1β) and IL-6 may play a central role in the acceleration of postmenopausal bone loss, but observational studies have led to contradictory results. Estrogen-dependent changes in the production of IL-1 receptor antagonist (IL-1ra) and the soluble IL-6 receptor (sIL-6R) potentially modify cytokine bioactivity. We therefore assessed the impact of menopause and hormone replacement therapy (HRT) on cytokines and activity modifiers in serum within a 5-year longitudinal study. One hundred sixty perimenopausal women (age 50.1 ± 2.8 years) were randomized to HRT or no treatment. Serum IL-6 increased with age (r = 0.16; p < 0.05), but cytokines did not correlate with baseline bone mineral density (BMD). HRT led to small increases in IL-1ra (p < 0.001) and IL-6 (p < 0.05), with a decrease in sIL-6R (p < 0.01) and no change in IL-1β. No changes were observed in the control group. IL-1ra was inversely correlated with bone loss at the ultradistal forearm (r = 0.29; p < 0.05) and to a lesser degree at the spine (r = 0.20; p = 0.09). In addition, there was a weak positive correlation between sIL-6R and bone loss at the ultradistal forearm (r = 0.26; p < 0.05). High IL-6 levels were associated with slower bone loss (spine r = 0.31, p < 0.01) and controlling for age did not diminish this association. The percent change in sIL-6R during HRT was correlated with the bone loss at the femoral neck (r = −0.29; p < 0.01) and weakly with bone loss in the spine (r = −0.16; p = 0.17). In conclusion, serum IL-1ra and sIL-6R are influenced by HRT and are associated with the rate of bone loss in perimenopausal women.
Partitioning a Daily Mechanical Stimulus into Discrete Loading Bouts Improves the Osteogenic Response to Loading
Authors: Robling Alexander G. , Burr David B. , Turner Charles H.
Published: April 2006

A single 3-minute bout of mechanical loading increases bone formation in the rat tibia. We hypothesized that more frequent, shorter loading bouts would elicit a greater osteogenic response than a single 3-minute bout. The right tibias of 36 adult female Sprague-Dawley rats were subjected to 360 bending cycles per day of a 54N force delivered in 1, 2, 4, or 6 bouts on each of the 3 loading days. Rats in the 6-bouts/day group received 60 bending cycles per bout (60 × 6); rats in the 4-bouts/day group received 90 bending cycles per bout (90 × 4); the 2- and 1-bouts/day groups received 180 and 360 bending cycles per bout, respectively (180 × 2 and 360 × 1). A nonloaded, age-matched control group (0 × 0) and two sham-bending groups (60 × 6 and 360 × 1) also were included. Fluorochrome labeling revealed a 10-fold increase in endocortical lamellar bone formation rate (BFR/bone surface [BS]) in the right tibia versus the left (nonloaded) side in the 60 × 6 bending group. Endocortical BFR/BS in the right tibia of the 4-, 2-, and 1-bout bending groups exhibited 8-, 4-, and 4-fold increases, respectively, over the control side. Relative (right minus left) values for endocortical BFR/BS, mineralizing surface (MS/BS), and mineral apposition rate (MAR) were 65–94% greater in the 90 × 4 and 60 × 6 bending groups compared to the 360 × 1 bending group. Sham-bending tibias exhibited relative endocortical bone formation values similar to those collected from the control (0 × 0) group. The data show that 360 daily loading cycles applied at intervals of 60 × 6 or 90 × 4 represent a more osteogenic stimulus than 360 cycles applied all at once, and that mechanical loading is more osteogenic when divided into discrete loading bouts. Presumably, bone cells become increasingly “deaf” to the mechanical stimulus as loading cycles persist uninterrupted, and by allowing a rest period between loading bouts, the osteogenic effectiveness of subsequent cycles can be increased.
In Vitro Apatite Induction by Phosphophoryn Immobilized on Modified Collagen Fibrils
Authors: Saito Takashi , Yamauchi Mitsuo , Abiko Yoshihiro , Matsuda Koichi , Crenshaw Miles A.
Published: April 2006

Noncollagenous phosphoproteins that interact with type I collagen are thought to nucleate the mineral phase to collagen network of mineralized tissues. Previously, we found that phosphophoryn cross-linked to type I collagen was an effective nucleator of apatite. Here, we investigated the potential role of collagen telopeptide structure on this nucleation. We used pepsin and sodium borohydride (NaBH4) to modify the telopeptide region and reducible cross-links in the collagen fibrils and determined the effect on mineral induction by phosphophoryn cross-linked to it. The amount of phosphophoryn cross-linked to NaBH4-reduced collagen fibrils was higher than that to intact (unmodified) collagen fibrils. However, the amount of phosphophoryn cross-linked to collagen that lacked the telopeptides (atelocollagen) was 25% of that cross-linked to intact collagen fibrils. Each preparation was incubated at 37°C in metastable calcium phosphate solutions that did not spontaneously precipitate. Apatite was induced by phosphophoryn cross-linked to intact collagen fibrils at 15.0 h whereas phosphophoryn cross-linked to reduced collagen fibrils induced apatite formation after 10.9 h. Enough phosphophoryn was cross-linked to atelocollagen to induce mineral formation, but it did not. The failure of the phosphophoryn-atelocollagen complex to nucleate mineral might have been caused by a cross-linking pattern in the helical portion of the collagen molecule that did not promote the growth of the calcium-phosphate clusters into nuclei. The present study indicates that the telopeptide domains of type I collagen play a role in the interaction with phosphophoryn, which is critical for the nucleation process.
Hedgehog Proteins Stimulate Chondrogenic Cell Differentiation and Cartilage Formation
Authors: Enomoto-Iwamoto Motomi , Nakamura Takashi , Aikawa Tomonao , Higuchi Yoshinobu , Yuasa Takahito , Yamaguchi Akira , Nohno Tsutomu , Noji Sumihare , Matsuya Tokuzo , Kurisu Kojiro , Koyama Eiki , Pacifici Maurizio , Dr. Iwamoto Masahiro
Published: April 2006

Sonic hedgehog (Shh) and Indian hedgehog (Ihh) are important regulators of skeletogenesis, but their roles in this complex multistep process are not fully understood. Recent studies have suggested that the proteins participate in the differentiation of chondrogenic precursor cells into chondrocytes. In the present study, we have tested this possibility more directly. We found that implantation of dermal fibroblasts expressing hedgehog proteins into nude mice induces ectopic cartilage and bone formation. Immunohistological and reverse-transcription polymerase chain reaction (RT-PCR) analyses revealed that the ectopic tissues derived largely if not exclusively from host cells. We found also that treatment of clonal prechondrogenic RMD-1 and ATDC5 cells in culture with Ihh or recombinant amino half of Shh (recombinant N-terminal portion of Shh [rShh-N]) induced their differentiation into chondrocytes, as revealed by cytoarchitectural changes, Alcian blue staining and proteoglycan synthesis. Induction of RMD-1 cell differentiation by Ihh or rShh-N was synergistically enhanced by cotreatment with bone morphogenetic protein 2 (BMP-2) but was blocked by cotreatment with fibroblast growth factor 2 (FGF-2). Our findings indicate that hedgehog proteins have the ability to promote differentiation of chondrogenic precursor cells and that their action in this process can be influenced and modified by synergistic or antagonist cofactors.
Coordinate Expression of Novel Genes During Osteoblast Differentiation
Authors: Seth Arun , Lee Barbara K. , Qi Shirley , Vary Calvin P. H.
Published: April 2006

To achieve new insights into the coordinate regulation of gene expression during osteoblast differentiation we utilized an approach involving global analysis of gene expression to obtain the identities of messenger RNAs (mRNAs) expressed using an established in vitro model of bone development. MC3T3-E1 osteoblast-like cells were induced to differentiate by the addition of β-glycerophosphate (β-GP) and ascorbic acid. RNA samples derived from induced and uninduced control MC3T3-E1 cells were used to prepare complementary DNA (cDNA) for serial analysis of gene expression (SAGE). A preliminary SAGE database was produced and used to prepare a hybridization array to further facilitate the characterization of changes in the expression levels of 92 of the SAGE-mRNA assignments after induction of osteoblast differentiation, specifically after 6 days and 14 days of ascorbate treatment. SAGE-array hybridization analysis revealed coordinate induction of a number of mRNAs including Rab24, calponin, and calcyclin. Levels of MSY-1, SH3P2, fibronectin, α-collagen, procollagen, and LAMP1 mRNAs, present at day 6 postinduction, were markedly reduced by day 14 postinduction. A number of unanticipated and potentially important developmental genes were identified including the transforming growth factor β (TGF-β) superfamily member Lefty-1. Lefty-1 transcript and translation product were found to be induced during the course of MC3T3-E1 cell differentiation. We present evidence, using transient transfection and antibody neutralization approaches, that Lefty-1 modulates the induction of alkaline phosphatase (ALP) after treatment of MC3T3-E1 cells with ascorbate and β-GP. These data should provide useful new information for future analysis of transcriptional events in osteoblast differentiation and mineralization.
Evidence for the Involvement of Two Pathways in Activation of Extracellular Signal-Regulated Kinase (Erk) and Cell Proliferation by Gi and Gq Protein-Coupled Receptors in Osteoblast-Like Cells
Authors: Caverzasio Joseph , Palmer Gaby , Suzuki Atsushi , Bonjour Jean-Philippe
Published: April 2006

The mechanisms by which Gi and Gq protein-coupled receptors mediate mitogenic signaling in osteoblast-like cells are unknown and were investigated in MC3T3-E1 cells using specific receptor agonists such as lysophosphatidic acid (LPA) and prostaglandin F2α (PGF2α). In contrast to their implication in epidermal growth factor (EGF) receptor tyrosine kinase signaling, the adaptor protein Shc, the Grb2/Sos complex, and the small G protein Ras were not involved in the activation of Erk induced by either LPA or PGF2α in MC3T3-E1 cells, suggesting that activation of Erk by Gi and Gq protein-coupled receptors is Ras independent in these cells. Using specific kinase inhibitors and kinetic analyses, we provide evidence for two distinct components in the activation of Erk by Gi and Gq protein-coupled receptors in MC3T3-E1 cells including an Src-like kinase-dependent pathway and a protein kinase C (PKC)-dependent mechanism. Functional analyses suggested that these two components are required for optimal DNA synthesis in response to LPA and PGF2α. These results suggest the implication of two pathways in the stimulation of Erk and cell replication by growth factors acting through Gi and Gq protein-coupled receptors in bone-forming cells.
Effects of Glucocorticoids on Tumor Necrosis Factor α-Dependent Activation of Nuclear Factor κB and Expression of the Intercellular Adhesion Molecule 1 Gene in Osteoblast-Like ROS17/2.8 Cells
Authors: Kurokouchi Kazutoshi , Kambe Fukushi , Kikumori Toyone , Sakai Tadahiro , Sarkar Devanand , Ishiguro Naoki , Iwata Hisashi , Seo Hisao
Published: April 2006

Recently, we showed that tumor necrosis factor α (TNF-α) stimulates expression of the intercellular adhesion molecule 1 (ICAM-1) and interleukin-6 (IL-6) genes through activation of p65-p50 heterodimer nuclear factor κB (NF-κB) in rat osteoblast-like ROS17/2.8 cells. In the present study, we investigated effects of a synthetic glucocorticoid, dexamethasone (Dex), on TNF-α-dependent activation of NF-κB and expression of the ICAM-1 gene. ROS17/2.8 cells were pretreated with Dex for 6 h and then exposed to TNF-α. Electrophoretic mobility shift assay (EMSA) revealed that TNF-α-dependent activation of NF-κB was almost completely suppressed by Dex treatment. Increase in ICAM-1 messenger RNA (mRNA) level by TNF-α also was markedly suppressed by Dex. Western blot and immunocytochemical analyses showed that Dex attenuated the TNF-α-induced nuclear translocation of p65. Treatment with protein synthesis inhibitor cycloheximide (CHX) reversed the Dex effect, indicating that Dex requires de novo protein synthesis for its action. Northern blot analysis revealed that Dex increased IκB-α mRNA level synergistically with TNF-α, whereas it decreased p65 mRNA level. The p105 and IκB-β mRNA levels were not altered by Dex. Consistent with the mRNA level, Dex increased the amount of IκB-α protein in the cytoplasm in either the presence or the absence of TNF-α. Considering a role of IκB to sequester NF-κB in the cytoplasm, it was suggested that an increase in IκB-α protein and the concomitant decrease in p65 synthesis account for the Dex-induced suppression of NF-κB activation in osteoblastic cells.
Evaluation of Changes in Trabecular Bone Architecture and Mechanical Properties of Minipig Vertebrae by Three-Dimensional Magnetic Resonance Microimaging and Finite Element Modeling
Authors: Borah B. , Dufresne T. E. , Cockman M. D. , Gross G. J. , Sod E. W. , Myers W. R. , Combs K. S. , Higgins R. E. , Pierce S. A. , Stevens M. L.
Published: April 2006

The study objective was to analyze the three-dimensional (3D) trabecular architecture and mechanical properties in vertebral specimens of young and mature Sinclair minipigs to assess the relative contribution of architecture to bone strength. We used 3D magnetic resonance microimaging (MRμI) and direct image analysis to evaluate a set of standard structural measurements and new architectural descriptors of trabecular bone in biopsy specimens from L2, L3, and L4 vertebrae (n = 16 in each group) from young (mean age, 1.2 years) and mature (mean age, 4.8 years) minipigs. The measurements included bone volume/tissue volume (BV/TV), marrow star volume (Ma.St.V), connectivity density (ConnD), and two new parameters, percent platelike trabeculae (% plate) and percent bone in the load direction (% boneLD). The % plate, calculated from surface curvature, allowed the delineation of plates from rods. The % boneLD quantified the percentage of bone oriented along the long axis of the vertebral body. We showed that 3D MRμI can detect the subtle changes in trabecular architecture between the two age groups. ConnD, star volume, % plate, % boneLD, and BV/TV were found to be more effective than the model-based, derived indices (trabecular thickness [Tb.Th], trabecular separation [Tb.Sp], and trabecular number [Tb.N]) in differentiating the structural changes. BV/TV, % plate, and % boneLD significantly increased (p < 0.05) in all three vertebral sites of the mature minipigs. The significant decrease in ConnD and star volume in the mature vertebra was consistent with the concurrent increase of platelike trabecular bone (p < 0.05). Overall, ConnD, star volume, % plate, and % boneLD provided a coherent picture of the architectural changes between the two age groups. Apparent modulus and maximum stress were determined experimentally on biopsy specimens from L2 vertebrae (n = 16). When apparent modulus was predicted using 3D MRμI data sets as input for finite element modeling (FEM), the results were similar to the experimentally determined apparent modulus (p = 0.12). Both methods were then used to compare the young and the mature animals; the experimental and predicted apparent modulus were significantly higher for the mature group (p = 0.003 and 0.012, respectively). The experimental maximum stress in the vertebra of the mature animals was twice as high as that for the young animals (p = 0.006). Bone quantity (BV/TV or bone mineral content [BMC]) alone could explain approximately 74–85% of the total variability in stress and modulus. The inclusion of either ConnD or % boneLD with BV/TV in a multiple regression analysis significantly improved the predictability of maximum stress, indicating that architecture makes additional contributions to compressive strength in normal minipig vertebra.
Effect of Calcitriol on Bone Loss After Cardiac or Lung Transplantation
Authors: Sambrook Philip , Henderson N. Kathy , Keogh Anne , MacDonald Peter , Glanville Allan , Spratt Phillip , Bergin Peter , Ebeling Peter , Eisman John
Published: April 2006

Rapid bone loss after cardiac and lung transplantation results in an increased risk of osteoporotic fracture. This study examined the efficacy of treatment with calcitriol (1,25-dihydroxyvitamin D3) in preventing bone loss in patients undergoing cardiac or lung transplantation. In this 2-year double-blind, stratified study, 65 patients undergoing cardiac or single lung transplantation were randomly allocated to receive either placebo or calcitriol (0.5-0.75 μg/day), the latter for either 12 months or 24 months. All patients received 600 mg calcium/day. Bone mineral density (BMD) was measured every 6 months for 2 years by dual-energy X-ray absorptiometry. There was no significant difference between groups with respect to age or cumulative dose of prednis(ol)one or cyclosporine over the 2 years. Bone loss at the proximal femur was significantly reduced or prevented at all three sites by treatment with calcitriol for 2 years compared with treatment with calcium alone. Treatment with calcitriol for 12 months followed by calcium for 12 months resulted in similar proximal femoral bone loss to that seen in those patients treated with calcium for 24 months, suggesting calcitriol prophylaxis needs to be continued beyond 12 months. At the lumbar spine, there were no significant differences in BMD between groups. Over a period of 2 years, 22 new vertebral fractures/deformities occurred in 4 patients treated with calcium alone compared with one new vertebral fracture in 1 patient treated with calcitriol. Because the sample size was too low to provide reliable interpretation of vertebral fracture rates, this difference is likely a chance result. Mild hypercalcemia was common with calcitriol therapy, as was mild hypercalciuria (59% of patients vs. 10% controls), but there were no significant differences between groups in serum creatinine after 2 years. These data suggest calcitriol has a role in reducing proximal femur bone loss after cardiac or lung transplantation but treatment needs to be continued beyond 1 year.
Serum Estradiol and Sex Hormone-Binding Globulin and the Risk of Hip Fracture in Elderly Women: The EPIDOS Study
Authors: Chapurlat Roland D. , Garnero Patrick , Bréart Gérard , Meunier Pierre J. , Delmas Pierre D.
Published: April 2006

It has been suggested that low serum 17β-estradiol (E2) and sex hormone-binding globulin (SHBG) may predict hip fracture in postmenopausal women. We have investigated the predictive value of serum E2 and SHBG concentrations and urinary deoxypyridinoline (D-Pyr) and type I collagen breakdown products (CTX) in a large prospective cohort of 7598 healthy elderly ambulatory women (EPIDOS study), aged 75 years or more. We performed a nested case control study, by matching 212 patients with incident hip fracture with 636 controls. Mean follow-up was 3.3 years (maximum, 4.9 years). Women having serum E2 below the limit of detection (3 pg/ml), that is, 2% of the population, were not at higher risk, with a relative hazard (RH) of 1.59 (95% CI = 0.45-5.55). Women having serum E2 below 5, 6, 7, or 8 pg/ml, in the lowest quartile, or below the median had no increased risk of hip fracture. In contrast, women having serum E2 in the highest quartile (i.e., ≥10 pg/ml) were protected, with an RH of 0.66 (0.44-0.98) that did not remain significant after adjustment for weight (RH = 0.71 [0.47-1.06]). High serum SHBG values with different cut-offs tended to be associated with an increased risk of hip fracture. Women in the highest quartile had an RH of 2.5 (1.37-4.61), compared with those in the lowest quartile, that decreased markedly after adjustment for body weight (1.61 [0.99-2.62]). The highest quartile of the ratio E2/SHBG, which is an index of free E2, was associated with a lower hip fracture risk (RH = 0.6 [0.4-0.91]) that was no longer significant after adjustment for weight. In contrast, urinary D-Pyr and CTX, when elevated above the upper limit of premenopausal values, were predictive of hip fracture, with an RH of 2.07 (1.49-2.9) and 1.67 (1.19-2.32), respectively, even after adjustment for body weight, serum E2, and SHBG. We conclude that in healthy elderly French women over 75 years of age, serum E2 and E2/SHBG in the highest quartile are associated with a lower risk of hip fracture and that this association is explained by a higher body weight. In addition, serum levels of E2 and SHBG do not account for the increased risk of hip fracture associated with high levels of bone resorption markers.
Effects of Oral Alendronate in Elderly Patients with Osteoporosis and Mild Primary Hyperparathyroidism
Authors: Rossini Maurizio , Gatti Davide , Isaia Giancarlo , Sartori Leonardo , Braga Vania , Adami Silvano
Published: April 2006

In a large proportion of the patients with primary hyperparathyroidism (PHPT), a variable degree of osteopenia is the only relevant manifestation of the disease. Low bone mineral density (BMD) in patients with PHPT is an indication for surgical intervention because successful parathyroidectomy results in a dramatic increase in BMD. However, low BMD values are almost an invariable finding in elderly women with PHPT, who are often either unwilling or considered unfit for surgery. Bisphosphonates are capable of suppressing parathyroid hormone (PTH)-mediated bone resorption and are useful for the prevention and treatment of postmenopausal osteoporosis. In this pilot-controlled study, we investigated the effects of oral treatment with alendronate on BMD and biochemical markers of calcium and bone metabolism in elderly women presenting osteoporosis and mild PHPT. Twenty-six elderly patients aged 67–81 years were randomized for treatment with either oral 10 mg alendronate on alternate-day treatment or no treatment for 2 years. In the control untreated patients a slight significant decrease was observed for total body and femoral neck BMD, without significant changes in biochemical markers of calcium and bone metabolism during the 2 years of observation. Urine deoxypyridinoline (Dpyr) excretion significantly fell within the first month of treatment with alendronate, while serum markers of bone formation alkaline phosphatase and osteocalcin fell more gradually and the decrease became significant only after 3 months of treatment; thereafter all bone turnover markers remained consistently suppressed during alendronate treatment. After 2 years in this group we observed statistically significant increases in BMD at lumbar spine, total hip, and total body (+8.6 ± 3.0%, +4.8 ± 3.9%, and +1.2 ± 1.4% changes vs. baseline mean ± SD) versus both baseline and control patients. Serum calcium, serum phosphate, and urinary calcium excretion significantly decreased during the first 3-6 months but rose back to the baseline values afterward. Increase in serum PTH level was statistically significant during the first year of treatment. These preliminary results may make alendronate a candidate as a supportive therapy in patients with mild PHPT who are unwilling or are unsuitable for surgery, and for whom osteoporosis is a reason of concern.
Bone Mineral Changes in Obese Women During a Moderate Weight Loss With and Without Calcium Supplementation
Authors: Jensen Lars Bjørn , Kollerup Gina , Quaade Flemming , SØRensen Ole Helmer
Published: April 2006

A significant relationship between body weight (BW) and bone mass (BM) has been established previously. A diet-induced weight loss is accompanied by a significant decrease in bone mineral density (BMD) and total body bone mineral (TBBM), but the underlying mechanisms are not clarified. Sixty-two obese women were included in the study. Dual-energy X-ray absorptiometry (DXA) and measurements of a series of calcium-regulating hormones and biochemical markers of bone turnover were performed at baseline and after 1 month and 3 months on a low calorie diet. Thirty of the women were randomized to a daily supplement of 1 g of calcium. After an additional 3 months without dietary prescriptions or calcium supplements, a subgroup of 48 subjects (24 from each group) were scanned again using DXA. There was a significant decrease in TBBM after 1 month and 3 months. A similar pattern was observed in the bone mineral content (BMC) of the lumbar spine in the patients who did not receive a calcium supplement, whereas no changes occurred in the supplemented group. The initial calcium supplementation seemed to protect against bone loss in the lumbar spine but not in the TBBM. In the nonsupplemented group, a statistically significant inverse correlation was found between the calcium/creatinine ratio in the morning urine and the changes in BMC of the lumbar spine. Such a relationship was not seen in the calcium-supplemented group. In the nonsupplemented group, no significant biochemical changes were observed, whereas a significant decrease in serum parathyroid hormone (PTH) was seen in the calcium-supplemented group. This might explain some of the protective effects of calcium supplementation on trabecular bone mass. We conclude that a diet-induced weight loss is accompanied by a generalized bone loss, which probably is explained mainly by a reduced mechanical strain on the skeleton. This loss can be partly inhibited by a high calcium intake. Therefore, a calcium supplementation should be recommended during weight loss, even if the diet contains the officially recommended amounts of calcium.
Jumping Improves Hip and Lumbar Spine Bone Mass in Prepubescent Children: A Randomized Controlled Trial
Authors: Fuchs Robyn K. , Bauer Jeremy J. , Snow Christine M.
Published: April 2006

Physical activity during childhood is advocated as one strategy for enhancing peak bone mass (bone mineral content [BMC]) as a means to reduce osteoporosis-related fractures. Thus, we investigated the effects of high-intensity jumping on hip and lumbar spine bone mass in children. Eighty-nine prepubescent children between the ages of 5.9 and 9.8 years were randomized into a jumping (n = 25 boys and n = 20 girls) or control group (n = 26 boys and n = 18 girls). Both groups participated in the 7-month exercise intervention during the school day three times per week. The jumping group performed 100, two-footed jumps off 61-cm boxes each session, while the control group performed nonimpact stretching exercises. BMC (g), bone area (BA; cm2), and bone mineral density (BMD; g/cm2) of the left proximal femoral neck and lumbar spine (L1-L4) were assessed by dual-energy X-ray absorptiometry (DXA; Hologic QDR/4500-A). Peak ground reaction forces were calculated across 100, two-footed jumps from a 61-cm box. In addition, anthropometric characteristics (height, weight, and body fat), physical activity, and dietary calcium intake were assessed. At baseline there were no differences between groups for anthropometric characteristics, dietary calcium intake, or bone variables. After 7 months, jumpers and controls had similar increases in height, weight, and body fat. Using repeated measures analysis of covariance (ANCOVA; covariates, initial age and bone values, and changes in height and weight) for BMC, the primary outcome variable, jumpers had significantly greater 7-month changes at the femoral neck and lumbar spine than controls (4.5% and 3.1%, respectively). In repeated measures ANCOVA of secondary outcomes (BMD and BA), BMD at the lumbar spine was significantly greater in jumpers than in controls (2.0%) and approached statistical significance at the femoral neck (1.4%; p = 0.085). For BA, jumpers had significantly greater increases at the femoral neck area than controls (2.9%) but were not different at the spine. Our data indicate that jumping at ground reaction forces of eight times body weight is a safe, effective, and simple method of improving bone mass at the hip and spine in children. This program could be easily incorporated into physical education classes.
Intermittently Administered Human Parathyroid Hormone(1–34) Treatment Increases Intracortical Bone Turnover and Porosity Without Reducing Bone Strength in the Humerus of Ovariectomized Cynomolgus Monkeys
Authors: Burr David B. , Hirano Toru , Turner Charles H. , Hotchkiss Charlotte , Brommage Robert , Hock Janet M.
Published: April 2006

Cortical porosity in patients with hyperparathyroidism has raised the concern that intermittent parathyroid hormone (PTH) given to treat osteoporotic patients may weaken cortical bone by increasing its porosity. We hypothesized that treatment of ovariectomized (OVX) cynomolgus monkeys for up to 18 months with recombinant human PTH(1–34) [hPTH(1–34)] LY333334 would significantly increase porosity in the midshaft of the humerus but would not have a significant effect on the strength or stiffness of the humerus. We also hypothesized that withdrawal of PTH for 6 months after a 12-month treatment period would return porosity to control OVX values. OVX female cynomolgus monkeys were given once daily subcutaneous (sc) injections of recombinant hPTH(1–34) LY333334 at 1.0 μg/kg (PTH1), 5.0 μg/kg (PTH5), or 0.1 ml/kg per day of phosphate-buffered saline (OVX). Sham OVX animals (sham) were also given vehicle. After 12 months, PTH treatment was withdrawn from half of the monkeys in each treatment group (PTH1-W and PTH5-W), and they were treated for the remaining 6 months with vehicle. Double calcein labels were given before death at 18 months. After death, static and dynamic histomorphometric measurements were made intracortically and on periosteal and endocortical surfaces of sections from the middiaphysis of the left humerus. Bone mechanical properties were measured in the right humeral middiaphysis. PTH dose dependently increased intracortical porosity. However, the increased porosity did not have a significant detrimental effect on the mechanical properties of the bone. Most porosity was concentrated near the endocortical surface where its mechanical effect is small. In PTH5 monkeys, cortical area (Ct.Ar) and cortical thickness (Ct.Th) increased because of a significantly increased endocortical mineralizing surface. After withdrawal of treatment, porosity in PTH1-W animals declined to sham values, but porosity in PTH5-W animals remained significantly elevated compared with OVX and sham. We conclude that intermittently administered PTH(1–34) increases intracortical porosity in a dose-dependent manner but does not reduce the strength or stiffness of cortical bone.
Resistance Training over 2 Years Increases Bone Mass in Calcium-Replete Postmenopausal Women
Authors: Kerr Deborah , Ackland Timothy , Maslen Barbara , Morton Alan , Prince Richard
Published: April 2006

Understanding the stress/strain relationship between exercise and bone is critical to understanding the potential benefit of exercise in preventing postmenopausal bone loss. This study examined the effect of a 2-year exercise intervention and calcium supplementation (600 mg) on bone mineral density (BMD) in 126 postmenopausal women (mean age, 60 ± 5 years). Assignment was by block randomization to one of three groups: strength (S), fitness (F), or nonexercise control (C). The two exercise groups completed three sets of the same nine exercises, three times a week. The S group increased the loading, while the F group had additional stationary bicycle riding with minimal increase in loading. Retention at 2 years was 71% (59% in the S group, 69% in the F group, and 83% in the C group), while the exercise compliance did not differ between the exercise groups (S group, 74 ± 13%; F group, 77 ± 14%). BMD was measured at the hip, lumbar spine, and forearm sites every 6 months using a Hologic 4500. Whole body BMD also was measured every 6 months on a Hologic 2000. There was no difference between the groups at the forearm, lumbar spine, or whole body sites. There was a significant effect of the strength program at the total (0.9 ± 2.6%; p < 0.05) and intertrochanter hip site (1.1 ± 3.0%; p < 0.01). There was a significant time and group interaction (p < 0.05) at the intertrochanter site by repeated measures. This study shows the effectiveness of a progressive strength program in increasing bone density at the clinically important hip site. We concluded that a strength program could be recommended as an adjunct lifestyle approach to osteoporosis treatment or used in combination with other therapies.
Tensile Stress Induces Bone Morphogenetic Protein 4 in Preosteoblastic and Fibroblastic Cells, Which Later Differentiate into Osteoblasts Leading to Osteogenesis in the Mouse Calvariae in Organ Culture
Authors: Ikegame Mika , Ishibashi Osamu , Yoshizawa Tatsuya , Shimomura Junko , Komori Toshihisa , Ozawa Hidehiro , Kawashima Hiroyuki
Published: April 2006

Mechanical stress is an important factor controlling bone remodeling, which maintains proper bone morphology and functions. However, the mechanism by which mechanical stress is transduced into biological stimuli remains unclear. Therefore, the purpose of this study is to examine how gene expression changes with osteoblast differentiation and which cells differentiate into osteoblasts. Tensile stress was applied to the cranial suture of neonatal mouse calvaria in a culture by means of helical springs. The suture was extended gradually, displaying a marked increase in cell number including osteoblasts. A histochemical study showed that this osteoblast differentiation began in the neighborhood of the existing osteoblasts, which can be seen by 3 h. The site of osteoblast differentiation moved with time toward the center of the suture, which resulted in an extension of osteoid. Scattered areas of the extended osteoid were calcified by 48 h. Reverse-transcription polymerase chain reaction (RT-PCR) revealed that tensile stress increased bone morphogenetic protein 4 (BMP-4) gene expression by 6 h and it remained elevated thereafter. This was caused by the induction of the gene in preosteoblastic cells in the neighborhood of osteoblasts and adjacent spindle-shaped fibroblastic cells. These changes were evident as early as 3 h and continued moving toward the center of the suture. The expression of Cbfa1/Osf-2, an osteoblast-specific transcription factor, followed that of BMP-4 and those cells positive with these genes appeared to differentiate into osteoblasts. These results suggest that BMP-4 may play a pivotal role by acting as an autocrine and a paracrine factor for recruiting osteoblasts in tensile stress-induced osteogenesis.
Immunosuppressant Use Without Bone Loss—Implications for Bone Loss After Transplantation
Authors: Goodman G. R. , Dissanayake I. R. , Sodam B. R. , Gorodetsky E. , Lu J. , Ma Y. F. , Jee W. S. S. , Epstein S.
Published: April 2006

Cyclosporine A (CsA) is associated with posttransplantation bone disease. Immunosuppressant drugs such as sirolimus (SRL), which are more potent and less deleterious than CsA, are being developed. Previous experiments have shown that SRL although immunosuppressive, is relatively bone sparing. The use of low doses of CsA and SRL in combination has displayed in vivo synergism. This study was initiated to examine the effect of low-dose CsA and SRL on bone metabolism, thereby hopefully providing a bone sparing immunosuppressive regimen for transplant recipients. One hundred and nineteen rats were divided into groups: basal, vehicle, CsA high dose, CsA low dose, SRL low dose, and combination low-dose CsA and SRL. The basal group was killed on day 0 for histomorphometry. The experimental groups were weighed and bled on days 0, 28, 56, and 84 and were killed on day 84 for histomorphometry. Serial assays for blood urea nitrogen (BUN), creatinine, and osteocalcin were performed. Osteocalcin was raised on days 28 and 56 in the high dose CsA group. Histomorphometry showed osteopenia with high-dose CsA. Low-dose CsA was relatively bone sparing, while low-dose SRL and combined low-dose CsA did not cause bone loss. In conclusion, the synergistic combination of low-dose CsA and SRL has the potential of providing both bone sparing and immunosuppressive benefits.