bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development and Characterization of Conditionally Immortalized Osteoblast Precursor Cell Lines from Human Bone Marrow Stroma
Authors: Hicok Kevin C. , Thomas Thierry , Gori Francesca , Rickard David J. , Spelsberg Thomas C. , Riggs B. Lawrence 
Published: April 2006

Abstract
Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312–324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34°C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5°C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5°C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1α,25-dihydroxyvitamin D3 (1,25(OH2)D3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10−8 M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10−8 M 1,25(OH2)D3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH2)D3 were similar to changes in mRNA levels. When cultured at 39.5°C with ascorbate and β-glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH2)D3, or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans.
Cyclic Stretch Enhances Gap Junctional Communication Between Osteoblastic Cells
Authors: Ziambaras Konstantinos , Lecanda Fernando , Steinberg Thomas H. , Civitelli Roberto 
Published: April 2006

Abstract
Mechanical loading is essential to maintain skeletal integrity. Because gap junctions in bone are affected by mechanical factors, we studied whether stretch, an anabolic stimulus for osteoblasts, modulates direct intercellular communication in these cells. Gap junctional communication during stretch was assessed using a newly developed method, the “parachute assay,” which allows monitoring of dye diffusion without disruption of the plasma membrane. Application of cyclic stretch for 2 or 24 h to well-coupled ROS 17/2.8 cells resulted in a 56.5% and 30.4% increase in dye coupling, respectively, compared with resting conditions. Stretch increased dye diffusion less dramatically (12.4% compared with unstimulated cells) in the poorly coupled UMR 106-01 cells. The stretch-induced increase of cell coupling was abolished in the presence of the gap junctional inhibitor, heptanol. Steady-state mRNA levels of connexin43 (Cx43), the gap junction protein that mediates cell-to-cell diffusion of negatively charged dyes between osteoblasts, were not different between control and stretched ROS 17/2.8 or UMR 106-01 cultures after various periods of cyclic stretch. However, phosphorylated forms of Cx43 protein were more abundant in stretched ROS 17/2.8 than in controls. This was associated with increased punctate Cx43-specific immunostain at appositional membranes of stretched cells. Thus, cyclic stretch increases gap junctional communication between osteoblastic cells by modulating intracellular localization of Cx43.
1,25(OH)2D3 Regulates Protein Kinase C Activity Through Two Phospholipid-Dependent Pathways Involving Phospholipase A2 and Phospholipase C in Growth Zone Chondrocytes
Authors: Sylvia V. L. , Schwartz Z. , Curry D. B. , Chang Z. , Dean D. D. , Boyan B. D. 
Published: April 2006

Abstract
We have previously shown that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in growth zone chondrocyte (GC) differentiation and that this effect is mediated by protein kinase C (PKC). The aim of the present study was to identify the signal transduction pathway used by 1,25(OH)2D3 to stimulate PKC activation. Confluent, fourth passage GC cells from costochondral cartilage were used to evaluate the mechanism of PKC activation. Treatment of GC cultures with 1,25(OH)2D3 elicited a dose-dependent increase in both inositol-1,4,5-trisphosphate and diacylglycerol (DAG) production, suggesting a role for phospholipase C and potentially for phospholipase D. Addition of dioctanoylglycerol to plasma membranes isolated from GCs increased PKC activity. Neither pertussis toxin nor choleratoxin had an inhibitory effect on PKC activity in control or 1,25(OH)2D3-treated GCs, indicating that neither Gi nor Gs proteins were involved. Phospholipase A2 inhibitors, quinacrine, OEPC (selective for secretory phospholipase A2), and AACOCF3 (selective for cytosolic phospholipase A2), and the cyclooxygenase inhibitor indomethacin decreased PKC activity, while the phospholipase A2 activators melittin and mastoparan increased PKC activity in GC cultures. Arachidonic acid and prostaglandin E2, two downstream products of phospholipase A2 action, also increased PKC activity. These results indicate that 1,25(OH)2D3-dependent stimulation of PKC activity is regulated by two distinct phospholipase-dependent mechanisms: production of DAG, primarily via phospholipase C and production of arachidonic acid via phospholipase A2.
Cathepsin K mRNA Detection Is Restricted to Osteoclasts During Fetal Mouse Development
Authors: Dr. Dodds R. A. , Connor J. R. , Drake F. , Feild J. , Gowen Maxine 
Published: April 2006

Abstract
We recently identified a novel cysteine protease, cathepsin K, by random sequencing of an osteoclast cDNA library, and in situ hybridization studies in adult human tissues demonstrated high and specific expression in osteoclasts. To determine whether the expression of cathepsin K mRNA during mouse embryogenesis was more widespread, cryostat sections of early (day 11–13) and late (day 15–17) mouse fetuses were analyzed by in situ hybridization. Serial cross-sections were collected through each fetus, and co-reacted for tartrate-resistant acid phosphatase (TRAP) and nonspecific esterase (NSE), selective markers for the osteoclast, and precursor cells derived from the macrophage/monocyte lineage, respectively. In the 11–13 day fetuses, cathepsin K mRNA was not expressed in any extraskeletal tissue; at this stage of embryogenesis, no osteoclasts are present. However, in the 15–17 day fetuses, a distinctive, developmental stage-dependent pattern of cathepsin K expression was observed in osteoclasts and preosteoclasts at sites of cartilage and bone modeling. Cathepsin K positive osteoclasts differentiated within a peripheral zone of the osteogenic stacked cell layer of the cartilage rudiments (prior to ossification), migrated and/or resorbed the bone collar, and invaded the cartilage core. Furthermore, following the invasive penetration of vasculature into the degenerating cartilage core, the calcified cartilage was resorbed by cathepsin K positive mononuclear osteoclast precursors (NSE+ve, negligible TRAP); cells positive for both enzymes were identified indicative of osteoclast differentiation. The deposition of bone by osteoblasts onto the cartilage remnants is followed by mononucleated and multinucleated osteoclastic resorption; these osteoclasts demonstrated intense cathepsin K expression. Similar expression patterns were observed at sites of intramembranous ossification. No expression was observed in chondrocytes, osteoblasts, marrow, or in any other nonskeletal tissue at these time points. These data indicated that cathepsin K expression during embryogenesis occurred only following the onset of osteoclast differentiation.
Wortmannin Inhibits Spreading and Chemotaxis of Rat Osteoclasts In Vitro
Authors: Pilkington Mary F. , Sims Stephen M. , Dr. Dixon S. Jeffrey 
Published: April 2006

Abstract
Wortmannin (WT) and 17β–hydroxywortmannin (HWT), which are inhibitors of phosphatidylinositol-3(OH)-kinase (PI3K), have been shown previously to inhibit bone resorption in vitro and in vivo, possibly by interfering with formation of the osteoclast ruffled border. Since migration of osteoclasts also plays an important role in the process of bone resorption, we investigated the effects of these inhibitors on osteoclast morphology and motility. Both HWT and WT caused a sustained decrease in the planar area of osteoclasts in vitro (half maximal effect at 25 and 165 nM, respectively), with the effect of HWT on cell area more readily reversible than WT. These agents also caused accumulation of intracellular vesicles. Time-lapse video microscopy was used to record the migration of osteoclasts in response to macrophage colony-stimulating factor (M-CSF) or vehicle, flowing passively from a micropipette positioned 200–400 μm from the cell. M-CSF caused directed migration of osteoclasts, indicating chemotaxis (over 3 h osteoclasts migrated 96 ± 14 μm in response to M-CSF vs. 11 ± 2 μm in control experiments). Both WT (100 or 500 nM) and LY294002 (100 μM), a specific PI3K inhibitor structurally unrelated to WT, significantly inhibited osteoclast chemotaxis in response to M-CSF. Taken together, these effects of WT, HWT, and LY294002 are consistent with an important role for PI3K in regulating cytoskeletal function in osteoclasts. The inhibitory effects of WT and HWT on bone resorption may be due, in part, to impairment of osteoclast motility.
Bone Mineral Density and Its Change in White Women: Estrogen and Vitamin D Receptor Genotypes and Their Interaction
Authors: Willing Marcia , Sowers Maryfran , Aron David , Clark M. K. , Burns Trudy , Bunten Carol , Crutchfield Mary , D'Agostino Danielle , Jannausch Mary 
Published: April 2006

Abstract
Low bone mineral density (BMD) is a major risk factor for development of osteoporosis; increasing evidence suggests that attainment and maintenance of peak bone mass as well as bone turnover and bone loss have strong genetic determinants. We examined the association of BMD levels and their change over a 3-year period, and polymorphisms of the estrogen receptor (ER), vitamin D receptor (VDR), type I collagen, osteonectin, osteopontin, and osteocalcin genes in pre- and perimenopausal women who were part of the Michigan Bone Health Study, a population-based longitudinal study of BMD. Body composition measurements, reproductive hormone profiles, bone-related serum protein measurements, and life-style characteristics were also available on each woman. Based on evaluation of women, ER genotypes (identified by PvuII [n = 253] and XbaI [n = 248]) were significantly predictive of both lumbar spine (p &lt; 0.05) and total body BMD level, but not their change over the 3-year period examined. The VDR BsmI restriction fragment length polymorphism was not associated with baseline BMD, change in BMD over time, or any of the bone-related serum and body composition measurements in the 372 women in whom it was evaluated. Likewise, none of the other polymorphic markers was associated with BMD measurements. However, we identified a significant gene × gene interaction effect (p &lt; 0.05) for the VDR locus and PvuII (p &lt; 0.005) and XbaI (p &lt; 0.05) polymorphisms, which impacted BMD levels. Women who had the (−/−) PvuII ER and bb VDR genotype combination had a very high average BMD, while individuals with the (−/−) PvuII ER and BB VDR genotype had significantly lower BMD levels. This contrast was not explained by differences in serum levels of osteocalcin, parathyroid hormone, 1,25-dihydroxyvitamin D, or 25-dihydroxyvitamin D. These data suggest that genetic variation at the ER locus, singly and in relation to the vitamin D receptor gene, influences attainment and maintenance of peak bone mass in younger women, which in turn may render some individuals more susceptible to osteoporosis than others.
Systemic Over-Secretion of Growth Hormone in Transgenic Mice Results in a Specific Pattern of Skeletal Modeling and Adaptation
Authors: Tseng Kuo-Fung , Goldstein Steven A. 
Published: April 2006

Abstract
The effects of growth hormone (GH) over-secretion on bone modeling and remodeling in transgenic mice were investigated using systematic and hierarchical methods. The results showed that continual systemic stimulation of GH over-secretion in mice resulted in an initial increase in bone mass, but its bone quality was not comparable with that of the littermate controls. Further continual over-stimulation of GH resulted in an increase in bone resorption leading to thinner cortices with inferior tissue mechanical properties. Three biological mechanisms following GH over-production are formulated to explain the observed results: GH over-secretion stimulated the production of inferior bone matrices, i.e., woven bone and cartilage, in addition to the production of normal lamellar bone; the increased bone formation stimulated by GH was followed by an exaggerated bone resorption process, resulting in cortical tissue with inferior mechanical properties; and a cortical shift occurred following GH over-stimulation that appeared to be caused by the combined effects of GH over-secretion and a variation in mechanical stimuli in the metallothionein-1 growth hormone regulating hormone transgenic mice. An “Optimal Strain Environment” model is proposed to help explain the observed results. From the results of the present and previous studies, we believe that the primary goal of bone adaptation to a biological perturbation, at least in mice, is to ensure proper mechanical function by maintaining structural integrity. In the process of maintaining a targeted whole bone structural integrity, the mechanical integrity of tissue may be sacrificed.
Collagen-Related Markers of Bone Turnover Reflect the Severity of Liver Fibrosis in Patients with Primary Biliary Cirrhosis
Authors: Guañabens Nuria , Parés Albert , Alvarez Luisa , De Osaba Ma Jesús Martínez , Monegal Ana , Peris Pilar , Ballesta Antonio M. , Rodés Joan 
Published: April 2006

Abstract
The influence of a nonskeletal disease with increased connective tissue synthesis or degradation in the collagen-related markers of bone turnover has been evaluated in 34 women with primary biliary cirrhosis (PBC; age range 41–81 years), a disease with increased hepatic fibrosis, often associated with osteoporosis. Serum osteocalcin (BGP), and carboxy-terminal (PICP) and amino-terminal (PINP) propeptides of type I collagen were assessed as indexes of bone formation, whereas serum tartrate-resistant acid phosphatase (TRAP), and cross-linked carboxy-terminal telopeptide of type I collagen (ICTP), and urinary hydroxyproline (HYP), pyridinoline (PYR), deoxypyridinoline (DPYR), and type I collagen cross-linked N- (NTX) and C-telopeptide (CTX) were measured as markers of bone resorption. The histologic stage of the disease and serum amino-terminal propeptide of type III collagen (PIIINP) as an index of liver fibrogenesis were also evaluated. BGP levels were significantly lower, whereas PICP and PINP levels were higher in patients than in controls. Among the bone resorption markers, serum ICTP and urinary PYR, DPYR, HYP, NTX, and CTX levels were significantly higher in patients than in controls. Serum PIIINP levels were also increased in PBC patients. BGP did not correlate with PICP and PINP, but these markers of bone formation as well as ICTP, PYR, DPYR, and NTX correlated with serum PIIINP levels. Serum TRAP did not correlate with collagen-related markers of bone resorption. Moreover, patients with PIIINP and bilirubin above normal levels had higher PICP, PINP, ICTP PYR, DPYR, CTX, and NTX. These markers correlated with the histologic stage of the disease, but not with osteopenia measured by densitometric procedures in 22 patients. In conclusion, collagen-related markers of bone turnover do not reflect bone remodeling in PBC. The close association of these markers with PIIINP and the clinical and histologic stage of the liver disease suggests that they are influenced by liver collagen metabolism.
Collagen Iα1 Sp1 Polymorphism, Bone Mass, and Bone Turnover in Healthy French Premenopausal Women: The OFELY Study
Authors: Garnero P. , Borel O. , Grant S. F. A. , Ralston S. H. , Delmas P. D. 
Published: April 2006

Abstract
Bone mineral density (BMD) is under strong genetic control. Recent work has suggested that a polymorphism affecting an Sp1 binding site in the collagen I (COLI) A1 gene is associated with BMD and vertebral fracture in postmenopausal women. We analyzed this polymorphism in relation to BMD and bone turnover in 220 healthy premenopausal women aged 31–57 years. There were 61% SS homozygotes, 35% Ss heterozygotes, and 4% ss homozygotes, genotype frequencies similar to those previously reported in other Caucasian populations. Women in the three genotype groups were matched for age, body weight, physical activity, smoking habits, and oral contraceptive use, but height was greatest in the SS group and lowest in the ss group (p = 0.03). Between-group comparisons by analysis of variance (ANOVA) showed that COLI A1 genotype was significantly associated with spine BMD (p = 0.05), total body BMD (p = 0.046), and total body bone mineral content (BMC) (p = 0.02), but the differences between extreme genotypes were small (4, 5, and 10%, for spine BMD, total body BMD, and total body BMC, respectively). After adjustment for height, the differences between genotypes decreased and were no longer significant by ANOVA (p = 0.08, 0.17, and 0.33 for spine BMD, total body BMD, and total body BMC). Furthermore, no significant difference between genotypes was observed for femoral neck, trochanter, Ward's triangle, or forearm BMD. COLI A1 genotype was associated with serum C-terminal extension propeptide of type I collagen (p = 0.04), with lowest levels in ss individuals, but not with any other marker of bone formation (osteocalcin, alkaline phosphatase, and type I collagen N-terminal extension propeptide) or bone resorption (urinary excretion of type I collagen C and N telopeptide breakdown products). The COLI A1 Sp1 polymorphism is associated with height, peak total body BMD and BMC, and spine BMD. The genotype-specific differences account for only a small proportion of variance in BMD at these sites and are not significant after adjustment for height, suggesting that part of the effect on bone mass may be due to differences in body size. Our data support the view that COLI A1 may be a candidate gene for regulation of bone mass, but our results must be treated with caution, in view of the small number of ss individuals, and will require confirmation in larger studies.
The Relationship Between Three-Dimensional Connectivity and the Elastic Properties of Trabecular Bone
Authors: Kinney J. H. , Ladd A. J. C. 
Published: April 2006

Abstract
A finite-element model was used to explore the relationship between connectivity density and the elastic modulus of trabecular bone. Six cubic specimens of trabecular bone, three prepared from human distal radii and three from L1 vertebrae, were imaged with synchrotron microtomography. The three-dimensional images were reconstructed into binary volumes of mineralized bone and soft tissue, and incorporated into the finite-element model. The relationship between three-dimensional connectivity and elastic modulus was explored by uniform thinning (atrophy) and thickening (recovery) of the trabecular bone. Though no functional relationship was found between connectivity and elastic modulus, there was a linear relationship, after a full cycle of atrophy and recovery, between the loss of elastic modulus and the overall loss of connectivity. The results indicate that recovery of mechanical function depends on preserving or restoring trabecular connectivity.
Evidence That Ibuprofen Antagonizes Selective Actions of Estrogen and Tamoxifen on Rat Bone
Authors: Sibonga Jean D. , Bell Norman H. , Turner Russell T. 
Published: April 2006

Abstract
Studies were performed to determine if the nonsteroidal anti-inflammatory drug ibuprofen alters bone and mineral metabolism in female rats. In experiment 1, four groups of growing rats underwent either sham operation or ovariectomy (OVX). One week later, controlled-release pellets with ibuprofen or placebo were implanted subcutaneously at the back of the neck. Following 3 weeks of treatment, rats were sacrificed and blood and bone samples were removed for serum assays and histomorphometric analysis. Body growth rate and the static cortical bone measurements made at the tibial diaphysis did not change in response to OVX. OVX, however, did increase radial bone growth, lowered serum 17β-estradiol, reduced uterine weight, and decreased the cancellous bone area of the tibial metaphysis in the rats. Ibuprofen did not alter serum 17β-estradiol or uterine weight but reduced radial bone growth as well as cancellous bone area of the tibial metaphysis in both sham-operated and OVX animals. In experiments 2 and 3, we tested the influence of ibuprofen on the effects of the tissue-selective estrogen agonist tamoxifen and of exogenous 17β-estradiol in the OVX rat. Ibuprofen completely blocked the effects of tamoxifen and partially blocked the effects of 17β-estradiol to prevent cancellous osteopenia. In contrast, ibuprofen did not influence the effects of tamoxifen and 17β-estradiol to reduce radial bone growth. Besides the skeletal effects, ibuprofen suppressed estrogen-induced uterine growth. Our data suggest that ibuprofen blocks selective estrogen receptor-mediated activities in the rat.