bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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 &lt;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

Abstract
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 &gt;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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.