bluuebunny/crossref_metadata_2025_split huggingface dataset:
Localizational Alterations of Calcium, Phosphorus, and Calcification-Related Organics Such as Proteoglycans and Alkaline Phosphatase During Bone Calcification
Authors: Hoshi Kazuto , Ejiri Sadakazu , Ozawa Hidehiro 
Published: April 2006

Abstract
To further approach the mechanisms of bone calcification, embryonic rat calvariae were observed at electron microscopic level by the means of fine structures and various cytochemical localizations, including nonspecific proteoglycan (PG) stained by cuprolinic blue (CB), decorin, chondroitin sulfate, hyaluronan, and alkaline phosphatase (ALP), as well as the elemental mapping of calcium (Ca) and phosphorus (P) by energy-filtering transmission electron microscopy (EFTEM). In the calvariae, calcification advanced as the distance from osteoblasts increased. Closer to the osteoblasts, the osteoid was marked by an abundance of CB-positive PGs around collagen fibrils. After crystallization within matrix vesicles, calcified nodules formed and expanded, creating a coherent calcified matrix. The sizes of CB-positive PG-like structures diminished as calcification proceeded. Although small CB-positive structures were accumulated in early stage-calcified nodules, they were localized along the periphery of larger calcified nodules. Cytochemical tests for decorin, chondroitin sulfate, and hyaluronan determined their presence in the areas around collagen fibrils of the osteoid, as well as in and around calcified nodules, whereas ALP was found in the matrix vesicles, as well as in and around the calcified nodules. Ca tended to localize at the PG sites, while P often mapped to the collagen fibril structures, in the uncalcified matrix. In contrast, Ca/P colocalization was visible in and around the calcified nodules, where ALP and smaller CB-positive structures were observed. The difference in the localization patterns of Ca and P in uncalcified areas may limit the local [Ca2+][PO43−] product, leading to the general inhibition of hydroxyapatite crystallization. The downsizing of CB-positive structures suggested enzymatic fragmentation of PGs. Such structural alterations would contribute to the preservation and transport of calcium. ALP possesses the ability to boost local phosphate anion concentration. Therefore, structurally altered PGs and ALP may cooperate in Ca/P colocalization, thus promoting bone calcification.
A Novel Cell Culture Model of Chondrocyte Differentiation During Mammalian Endochondral Ossification
Authors: Cheung J. O. P. , Hillarby M. C. , Ayad S. , Hoyland J. A. , Jones C. J. P. , Denton J. , Thomas J. T. , Wallis G. A. , Grant M. E. 
Published: April 2006

Abstract
Endochondral ossification (EO) occurs in the growth plate where chondrocytes pass through discrete stages of proliferation, maturation, hypertrophy, and calcification. We have developed and characterized a novel bovine cell culture model of EO that mirrors these events and will facilitate in vitro studies on factors controlling chondrocyte differentiation. Chondrocytes derived from the epiphyses of long bones of fetal calves were treated with 5-azacytidine (aza-C) for 48 h. Cultures were maintained subsequently without aza-C and harvested at selected time points for analyses of growth and differentiation status. A chondrocytic phenotype associated with an extensive extracellular matrix rich in proteoglycans and collagen types II and VI was observed in aza-C-treated and -untreated cultures. aza-C-treated cultures were characterized by studying the expression of several markers of chondrocyte differentiation. Parathyroid hormone-related protein (PTHrP) and its receptor, both markers of maturation, were expressed at days 5–9. Type X collagen, which is restricted to the stage of hypertrophy, was expressed from day 11 onward. Hypertrophy was confirmed by a 14-fold increase in cell size by day 15 and an increased synthesis of alkaline phosphatase during the hypertrophic period (days 14–28). The addition of PTHrP to aza-C-treated cultures at day 14 led to the down-regulation of type X collagen by 6-fold, showing type X collagen expression is under the control of PTHrP as in vivo. These findings show that aza-C can induce fetal bovine epiphyseal chondrocytes to differentiate in culture in a manner consistent with that which occurs during the EO process in vivo.
Antagonism of the Osteoclast Vitronectin Receptor with an Orally Active Nonpeptide Inhibitor Prevents Cancellous Bone Loss in the Ovariectomized Rat
Authors: Lark Michael W. , Stroup George B. , Dodds Robert A. , Kapadia Rasesh , Hoffman Sandra J. , Hwang Shing Mei , James Ian E. , Lechowska Beata , Liang Xiaoguang , Rieman David J. , Salyers Kevin L. , Ward Keith , Smith Brian R. , Miller William H. , Huffman William F. , Gowen Maxine 
Published: April 2006

Abstract
An orally active, nonpeptide Arg-Gly-Asp (RGD) mimetic αvβ3 antagonist, (S)-3-Oxo-8-[2-[6-(methylamino)pyridin-2-yl]-1-ethoxy]-2-(2,2,2-trifluoroethyl)-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetic acid (compound 1), has been generated, which prevented net bone loss and inhibited cancellous bone turnover in vivo. The compound binds αvβ3 and the closely related integrin αvβ5 with low nanomolar affinity but binds only weakly to the related integrins αIIbβ3, and α5β1. Compound 1 inhibited αvβ3-mediated cell adhesion with an IC50 = 3 nM. More importantly, the compound inhibited human osteoclast-mediated bone resorption in vitro with an IC50 = 11 nM. In vivo, compound 1 inhibited bone resorption in a dose-dependent fashion, in the acute thyroparathyroidectomized (TPTX) rat model of bone resorption with a circulating EC50 ∼ 20 μM. When dosed orally at 30 mg/kg twice a day (b.i.d.) in the chronic ovariectomy (OVX)-induced rat model of osteopenia, compound 1 also prevented bone loss. At doses ranging from 3 to 30 mg/kg b.i.d., compound 1 partially prevented the OVX-induced increase in urinary deoxypyridinoline. In addition, the compound prevented the OVX-induced reduction in cancellous bone volume (BV), trabecular number (Tb.N), and trabecular thickness (Tb.Th), as assessed by quantitative microcomputerized tomography (μCT) and static histomorphometry. Furthermore, both the 10-mg/kg and 30-mg/kg doses of compound prevented the OVX-induced increase in bone turnover, as measured by percent osteoid perimeter (%O.Pm). Together, these data indicate that the αVβ3 antagonist compound 1 inhibits OVX-induced bone loss. Mechanistically, compound 1 prevents bone loss in vivo by inhibiting osteoclast-mediated bone resorption, ultimately preventing cancellous bone turnover.
T Cell Activation Induces Human Osteoclast Formation via Receptor Activator of Nuclear Factor κB Ligand-Dependent and -Independent Mechanisms
Authors: Weitzmann M. Neale , Cenci Simone , Rifas Leonard , Haug Jeffrey , Dipersio John , Pacifici Roberto 
Published: April 2006

Abstract
In unstimulated conditions, osteoclast (OC) formation is regulated by stromal cell production of the key osteoclastogenic factors receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). However, the mechanisms of accelerated osteoclastogenesis and bone loss characteristic of inflammatory conditions are poorly understood but appear to involve T cells. In addition, the mechanism by which OCs arise spontaneously in cultures of peripheral blood mononuclear cells in the absence of stromal cells or added cytokines remains unclear. Using a stromal cell free human osteoclast generating system, we investigated the ability of activated T cells to support osteoclastogenesis. We show that when activated by phytohemagglutinin-P (PHA), T cells (both CD4+ and CD8+) stimulate human OC formation in vitro. Although both soluble M-CSF and RANKL were detected in activated T cell supernatants, the presence of M-CSF was not essential for macrophage survival or RANKL-dependent osteoclast formation, suggesting that other soluble T cell-derived factors were capable of substituting for this cytokine. We also found that saturating concentrations of osteoprotegerin (OPG) failed to neutralize 30% of the observed OC formation and that T cell conditioned medium (CM) could superinduce osteoclastogenesis in cultures of purified monocytes maximally stimulated by RANKL and M-CSF. Together, these data suggest that activated T cells support osteoclastogenesis via RANKL-dependent and -independent mechanisms. Although not relevant for T cell-induced osteoclastogenesis, secretion of soluble M-CSF is a previously undescribed property of activated T cells.
Efficacy of Etanercept for Wear Debris-Induced Osteolysis
Authors: Childs Lisa M. , Goater J. Jeffrey , O'Keefe Regis J. , Schwarz Edward M. 
Published: April 2006

Abstract
A major limitation of total joint arthroplasty is that up to 20% of patients require revision surgery to correct prosthetic loosening. Aseptic loosening is believed to result from the phagocytosis of wear debris particles by macrophages, which secrete proinflammatory cytokines that stimulate osteolysis. Tumor necrosis factor α (TNF-α) has been shown to be one of the prominent cytokines in this cascade and to be involved critically in the generation of particle-induced osteolysis. Etanercept is a soluble inhibitor of TNF-α, which is widely used for the treatment of rheumatoid arthritis. Here, we show this agent's ability to prevent wear debris-induced osteolysis. In vitro we show that Etanercept can inhibit directly osteoclastic bone resorption in a bone wafer pit assay, as well as cytokine production from titanium (Ti)-stimulated macrophages. Using a quantitative in vivo model of wear debris-induced osteolysis, we show that Etanercept prevents bone resorption and osteoclastogenesis. In mice treated with Etanercept at the time of osteolysis induction, bone resorption and osteoclast numbers were reduced to background levels in both normal and human TNF-α (hTNF-α) transgenic mice. In an effort to evaluate its effect on established osteolysis, Etanercept was administered 5 days after Ti implantation, and we observed that further osteolysis was prevented. These data support the concept that TNF-α is involved critically in osteoclastogenesis and bone resorption during periprosthetic osteolysis and suggest that soluble TNF-α inhibitors may be useful as therapeutic agents for the treatment of prosthetic loosening in humans.
The Influence of Growth Hormone Deficiency, Growth Hormone Replacement Therapy, and Other Aspects of Hypopituitarism on Fracture Rate and Bone Mineral Density
Authors: Wüster Christian , Abs Roger , Bengtsson Bengt-Åke , Bennmarker Helge , Feldt-Rasmussen Ulla , Hernberg-Ståhl Elizabeth , Monson John P. , Westberg Bjørn , Wilton Patrick 
Published: April 2006

Abstract
To assess the influence of factors affecting fracture risk and bone density in adult hypopituitary patients with growth hormone deficiency (GHD), data from a large-scale pharmacoepidemiological survey (the Pharmacia & Upjohn International Metabolic Database [KIMS]) were analyzed and compared with data from a control population (the European Vertebral Osteoporosis Study [EVOS]). The KIMS group consisted of 2084 patients (1112 men and 972 women) with various types of pituitary disease and EVOS consisted of 1176 individuals (581 men and 595 women). Fracture and bone mineral density (BMD) data were available from 2024 patients from the KIMS group and 392 patients from EVOS. The prevalence of fractures in patients with hypopituitarism was 2.66 times that in the non-GH-deficient EVOS population. Adult-onset hypopituitarism with GHD was associated with a higher fracture risk than childhood-onset disease, and patients with isolated GHD had a similar prevalence of fractures to those with multiple pituitary hormone deficiencies. Hormonal replacement therapy with L-thyroxine, glucocorticoids, and sex steroids did not affect the risk of fracture in KIMS patients. In addition, fracture rates in KIMS were independent of body mass index (BMI) and the country of origin. However, smoking was associated with a higher fracture rate in this group. In summary, this is the first large-scale analysis to support the hypothesis of an increased fracture risk in adult patients with hypopituitarism and GHD. This increased risk appears to be attributable to GHD alone, rather than to other pituitary hormone deficiencies or to their replacement therapy.
Long-Term Effect of Incadronate Disodium (YM-175) on Fracture Healing of Femoral Shaft in Growing Rats
Authors: Li Chaoyang , Mori Satoshi , Li Jiliang , Kaji Yoshio , Akiyama Tomoyuki , Kawanishi Jun , Norimatsu Hiromichi 
Published: April 2006

Abstract
The aim of this study was to investigate the long-term effect of incadronate on fracture healing of the femoral shaft in rats. Female Sprague-Dawley 8-week-old rats were injected subcutaneously (sc) with either vehicle (V group) or two doses of incadronate (10 μg/kg and 100 μg/kg) three times a week for 2 weeks. Right femoral diaphysis was then fractured and fixed with intramedullary stainless wire. Just after fracture, incadronate treatment was stopped in pretreatment groups (P groups: P-10 and P-100) or continued in continuous treatment groups (C groups: C-10 and C-100). All rats were killed at 25 weeks or 49 weeks after surgery. Fractured femur was evaluated radiologically and mechanically and then stained in Villanueva bone stain and embedded in methyl methacrylate. Undecalcified cross-sections from the fracture area were evaluated microradiologically and histomorphometrically. Radiographic observation showed that the fracture line disappeared in all groups. Cross-sectional area in the C-100 group was the biggest among all groups and in the C-10 group was larger than that in the V group at 25 weeks. Histological and histomorphometric observations showed that the process of fracture healing was delayed under continuous treatment with incadronate as evidenced by the delay of both lamellar cortical shell formation and resolution of original cortex in C groups. Percent linear labeling perimeter, mineral apposition rate (MAR), and bone formation rate (BFR) in C groups significantly decreased compared with the other groups, indicating that the callus remodeling was suppressed under continuous treatment, especially with a high dose. Mechanical study showed that the stiffness and ultimate load of the fractured femur in the C 100 group were the highest among all groups at both 25 weeks and 49 weeks. In conclusion, this study showed that long-term continuous treatment with incadronate delayed the process of fracture healing of femur in rats, especially under high dose but it did not impair the recovery of mechanical integrity of the fracture.
Mechanical Consequences of Bone Loss in Cancellous Bone
Authors: Van Der Linden J. C. , Homminga J. , Verhaar J. A. N. , Weinans H. 
Published: April 2006

Abstract
The skeleton is continuously being renewed in the bone remodeling process. This prevents accumulation of damage and adapts the architecture to external loads. A side effect is a gradual decrease of bone mass, strength, and stiffness with age. We investigated the effects of bone loss on the load distribution and mechanical properties of cancellous bone using three-dimensional (3D) computer models. Several bone loss scenarios were simulated. Bone matrix was removed at locations of high strain, of low strain, and random throughout the architecture. Furthermore, resorption cavities and thinning of trabeculae were simulated. Removal of 7% of the bone mass at highly strained locations had deleterious effects on the mechanical properties, while up to 50% of the bone volume could be removed at locations of low strain. Thus, if remodeling would be initiated only at highly strained locations, where repair is likely needed, cancellous bone would be continuously at risk of fracture. Thinning of trabeculae resulted in relatively small decreases in stiffness; the same bone loss caused by resorption cavities caused large decreases in stiffness and high strain peaks at the bottom of the cavities. This explains that a reduction in the number and size of resorption cavities in antiresorptive drug treatment can result in large reductions in fracture risk, with small increases in bone mass. Strains in trabeculae surrounding a cavity increased by up to 1000 microstrains, which could lead to bone apposition. These results give insight in the mechanical effects of bone remodeling and resorption at trabecular level.
Caspase-Dependent Cleavage of Cadherins and Catenins During Osteoblast Apoptosis
Authors: Hunter Irene , Mcgregor Duncan , Robins Simon P. 
Published: April 2006

Abstract
As transmembrane, Ca2+-dependent cell-cell adhesion molecules, cadherins play a central role in tissue morphogenesis and homeostasis. Stable adhesion is dependent on interactions of the cytoplasmic domain of the cadherins with a group of intracellular proteins, the catenins. In the present study, we have detected the expression of α-, β-, and γ-catenins in human osteoblasts, which assemble with cadherins to form two distinct complexes containing cadherin and α-catenin, with either β- or γ-catenin. In osteoblasts undergoing apoptosis, proteolytic cleavage of N-cadherin and β- and γ- catenins but not α-catenin was associated with the activation of caspase-3 and prevented by the caspase inhibitor Z-VAD-fmk. The pattern of cadherin/catenin cleavage detected in apoptotic osteoblasts was reproduced in vitro by recombinant caspase-3. The presence of a 90-kDa extracellular domain fragment of N-cadherin in conditioned medium from apoptotic cells indicates that additional extracellular or membrane-associated proteases also are activated. Disruption of N-cadherin-mediated cell-cell adhesion with function-blocking antibodies induced osteoblast apoptosis, activation of caspases, and cleavage of β-catenin. These findings provide compelling evidence that N-cadherin-mediated cell-cell adhesion promotes osteoblast survival and suggest that the underlying mechanism may involve activation of β-catenin signaling.
Epigenetic Regulation of Human Bone Morphogenetic Protein 6 Gene Expression in Prostate Cancer
Authors: Tamada Hiroshi , Kitazawa Riko , Gohji Kazuo , Kitazawa Sohei 
Published: April 2006

Abstract
Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor-β (TGF-β) superfamily, are multifunctional molecules that regulate bone induction and organ development. Among BMPs, BMP-6 has been shown to be overexpressed in prostate cancer and is speculated to be associated with bone-forming skeletal metastasis. We investigated the regulatory mechanism of the BMP-6 gene expression in prostate cancer cell lines DU-145, LNCaP, PC-3, and PC-3M with regard to the methylation status of the CpG island in the 5′ flanking region of the human BMP-6 gene. By sequence-specific analysis of methylated cytosines, we show here that the methylation status of the CpG loci around the Sp1 site of the BMP-6 promoter is related to its steady-state expression and an alternative splicing of messenger RNA (mRNA) in prostate cancer cell lines. Furthermore, a study of clinical cases of benign and malignant prostate lesion by in situ hybridization showed that BMP-6 expression was high at both primary and secondary sites in cases of advanced cancer with metastasis. Demethylation of the CpG loci around the Sp1 binding site was shown in cases with high BMP-6 expression by sequencing analysis of the methylated cytosine from paraffin-embedded materials. Our results suggested that during cancer progression, besides inactivation of tumor suppressor genes by hypermethylation, activation of certain genes like BMP-6 by selective demethylation was a common epigenetic event giving a variable character to the invading and metastasizing cancer cells.
Regulation of the 1b Isoform of the Plasma Membrane Calcium Pump by 1,25-Dihydroxyvitamin D3 in Rat Osteoblast-Like Cells
Authors: Glendenning Paul , Ratajczak Thomas , Dick Ian M. , Prince Richard L. 
Published: April 2006

Abstract
The first isogene of the plasma membrane calcium pump (PMCA1) is expressed on the apical plasma membrane of osteoblasts, but its regulation by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] has not been studied in this cell type. We studied 1,25(OH)2D3 effects on PMCA1 function, protein, messenger RNA (mRNA), and isoform expression in osteoblasts. Of seven rat and human immortalized osteoblast-like cell lines studied, PMCA1 mRNA expression was confirmed in all. Only ROS 17/2.8 cells expressed measurable PMCA1 protein by Western analysis. Immunocytochemistry indicated that PMCA1 was expressed predominantly on the plasma membrane of ROS 17/2.8 cells. The 1,25(OH)2D3 but not 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] treatment of confluent ROS 17/2.8 cells resulted in an approximate 3- to 5-fold dose-dependent increase in PMCA1 expression of message and protein as assessed by Western and Northern analysis and vesicular45Ca uptake of membrane vesicles. 1,25(OH)2D3 had no effect on PMCA1 posttranscriptional splicing. The 1b isoform of PMCA was expressed under all experimental conditions. 1,25(OH)2D3 favored increased expression of the 5.5 kilobases (kb) over the 7.5-kb PMCA1b transcript, with a 2-fold proportional increase in the smaller transcript relative to the larger transcript evident at the highest dose of 1,25(OH)2D3 studied. The resultant proportional increase in the smaller 5.5-kb transcript may increase mRNA stability and account for the increase in PMCA1b protein and function with 1,25(OH)2D3. These data provide evidence for the role of 1,25(OH)2D3 and PMCA1b in the regulation of calcium transport in bone cells.
Nitric Oxide Synthase Isoforms During Fracture Healing
Authors: Zhu Wei , Diwan Ashish D. , Lin Jian-Hao , Murrell George A. C. 
Published: April 2006

Abstract
We have shown previously that nitric oxide (NO) has regulatory effects on fracture healing. Our aim here was to investigate the temporal expression patterns of the three NO synthase (NOS) isoforms that are responsible for the generation of NO by semiquantitative competitive polymerase chain reaction (PCR) and immunoblot analysis after femoral fractures in rats. We found that 4 days after fracture, there were increases in the levels of messenger RNA (mRNA) for all three NOS isoforms, with peaks for the inducible NOS (iNOS; 35-fold increase, p &lt; 0.05) at day 4, the endothelial NOS (eNOS; 5-fold increase, p &lt; 0.05) at day 7, and the neuronal NOS (bNOS; 16-fold increase, p &lt; 0.05) at day 21. At a protein level, the time course expression of NOS isoforms was consistent with the results of those at the mRNA level. In addition, we have previously reported a 2.5-fold increase in NOS activity detected by [3H]arginine to [3H]citrulline conversion at day 15 compared with that at day 4 after fracture. The findings that the expression of NOS isoforms during fracture healing is type specific and time dependent are important and may have clinical applications in the regulation of bone repair by NOS inhibitors or stimulators at different stages after injury.