bluuebunny/crossref_metadata_2025_split huggingface dataset:
Establishing an Immortalized Human Osteoprecursor Cell Line: OPC1
Authors: Winn Shelley R. , Randolph Gannon , Uludag Hasan , Wong Shou C. , Hair Gregory A. , Hollinger Jeffrey O. 
Published: April 2006

Abstract
The present studies evaluated the feasibility of establishing a conditionally immortalized osteoprecursor cell line derived from human fetal bone tissue. Primary cultures were transfected with a plasmid in which the Mx-1 promoter drives the expression of SV40 T-antigen when activated by human A/D interferon. Several neomycin (G418)-resistant colonies were characterized for cell growth and alkaline phosphatase (ALP) enzyme activity. The clone, designated OPC1 (osteoblastic precursor cell line 1), which exhibited the highest ALP enzyme activity at passage 10 (P10), was selected for additional osteogenic phenotypic characterization. Reverse transcription-polymerase chain reaction (RT-PCR) phenotyping revealed abundant mRNA for osteocalcin (OC), osteonectin (ON), osteopontin (OP), parathyroid hormone receptor (PTHr), ALP, and procollagen type I (ProI). In addition, the levels of quantitative RT-PCR product of ON, OP, PTHr, and ProI mRNAs exhibited a marked up-regulation when maintained in medium containing an osteogenic supplement (OS). The ability to stimulate osteogenic differentiation was characterized in postconfluent OPC1 cells maintained in tissue culture medium supplemented with recombinant human bone morphogenetic protein-2 (rhBMP-2) either with or without an OS. All treatment groups exhibited a striking up-regulation of ALP enzyme activity that coincided with ALP histochemical observations. Postconfluent cells also exhibited the ability to form mineralized nodules under all treatments (confirmed by von Kossa histochemical staining and calcium deposition). An enzyme immunosorbent assay (EIA) was utilized to measure intact human OC from the OPC1 line under the various treatments. Abundant OC was evident in the tissue culture medium indicating de novo sythesis and release from the OPC1 line under appropriate conditions. The clonal human-derived OPC1 line represents a homogeneous osteogenic cell line that not only has maintained a consistent bone phenotype from P10 to at least P30, but has also exhibited the capacity to generate programmed differentiation in the presence of low dose rhBMP-2 (10 ng/ml). Thus, the OPC1 line is a human-derived osteoprecursor that provides a sensitive in vitro cell culture system to evaluate bone development, cell/biomaterial interactions, and may be a useful screen for putative bone differentiating factors.
The Transcription Factor Sox9 Is Involved in BMP-2 Signaling
Authors: Zehentner Barbara K. , Dony Carola , Burtscher Helmut 
Published: April 2006

Abstract
We investigated the regulation of Sox9, a transcription factor known to play a role in chondrogenesis, by bone morphogenetic protein-2 (BMP-2) and hedgehog proteins in order to better understand their signaling function in endochondral bone formation. The mesenchymal progenitor cell line C3H10T1/2 was stimulated with BMP-2. Sox9 expression levels were measured by quantitative reverse transcriptase-polymerase chain reaction and Northern analysis. We found that Sox9 was up-regulated by BMP-2 in a dose-dependent manner. The expression of Col2a1, a downstream response gene of Sox9, was also significantly increased upon BMP-2 addition. We also monitored Sox9 expression after the addition of BMP-2 to osteosarcoma cell lines; BMP-2 treatment increased Sox9 mRNA levels in MG63, considered to be early osteoblast-like, but not in human osteogenic sarcoma (HOS) cells, which are thought to be more advanced in the osteoblastic lineage. This response seems to be influenced by differences in BMP receptor expression; MG63 cells express BMP receptor IA (BMPR-IA), whereas HOS cells express BMPR-IA and BMPR-IB. We also saw an increase in Sox9 mRNA levels in BMP-2–treated primary human bone cells (HBCs) derived from femoral heads. We found that in addition to BMP-2, Sonic and Indian hedgehog can increase Sox9 expression in C3H10T1/2 and primary HBCs. Time course studies with C3H10T1/2 cells after BMP-2 stimulation showed increasing expression of cartilage markers, decrease of collagen I mRNA, and a late induction of osteocalcin expression. Moreover, the treatment of C3H10T1/2 cells with Sox9 antisense oligonucleotides revealed that Sox9 is a downstream mediator of BMP-2 affecting the expression of chondrocyte and osteoblast marker genes. Our data show that Sox9 is an important downstream mediator of the BMP-2 and hedgehog signaling pathways in osteogenic cells.
Mechanical Strain Stimulates ROS Cell Proliferation Through IGF-II and Estrogen Through IGF-I
Authors: Cheng Ming Zhao , Zaman Gul , Rawlinson Simon C. F. , Mohan Subburaman , Baylink David J. , Lanyon Lance E. 
Published: April 2006

Abstract
The mechanism by which mechanical strain stimulates bone cell proliferation was investigated and compared with that of estrogen in ROS 17/2 .8 cells. Similarity of strain-related responses between ROS cells and osteoblasts was established by demonstrating that ROS cells respond to a short single period of strain in their substrate (1000–3500 μϵ, 600 cycles, 1 Hz) by a similar strain magnitude-related increase in glucose 6-phosphate dehydrogenase activity as rat osteoblasts and osteocytes in explants in situ. ROS17/2 .8 cells also showed similar proliferative responses to strain and 17β-estradiol, as assessed by [3H]thymidine incorporation and cell counting, as primary cultures of long bone-derived osteoblast-like cells. Strain-related increase in proliferation in ROS cells was accompanied by a 4-fold increase in levels of insulin-like growth factor-II (IGF-II) in conditioned medium. Neither strain nor estrogen had an effect on the conditioned medium levels of IGF-I. Exogenous truncated IGFs tIGF-I and tIGF-II both increased proliferation in a dose-dependent manner. The neutralizing monoclonal antibody (nMAb) to IGF-I blocked proliferation stimulated by tIGF-I but not that due to tIGF-II and vice versa. IGF-I receptor blocking antibody (IGF-IRBAb) blocked the proliferative effect of tIGF-I but not that to tIGF-II. The proliferative effect of estrogen was abolished by IGF-I nMAb and IGF-IRBAb, but these antibodies had no effect on the proliferative response to strain. In contrast IGF-II nMAb abolished the proliferative effect of strain but had no effect on that of estrogen. These data show that ROS17/2 .8 cells have similar responses to strain and estrogen qualitatively and quantitatively as rat osteoblasts in situ and rat long bone–derived osteoblast-like cells in primary culture. Estrogen-related proliferation in ROS17/2 .8 cells appears to be mediated by IGF-I acting through the IGF-I receptor and does not involve IGF-II. In contrast, strain-related proliferation appears to be mediated by IGF-II and does not involve either IGF-I or the IGF-I receptor.
Bone Physiology During Pregnancy and Lactation in Young Macaques
Authors: Ott Susan M. , Lipkin Edward W. , Newell-Morris Laura 
Published: April 2006

Abstract
We used a nonhuman primate model (Macaca nemestrina) of adolescent human pregnancy to characterize bone remodeling at midpregnancy and at weaning and the associated changes in bone mass. In this longitudinal study, 125 nulliparous females were followed through pregnancy, 6 months of lactation, and 3 months postweaning; 13 nonpregnant females served as controls. Between early pregnancy and midpregnancy, the whole body bone mineral increased. There was no significant change between midpregnancy and parturition. Between parturition and 3 months lactation, the animals lost 3.0% of their bone mineral (p &lt; 0.01), which was regained by 3 months after weaning. The vertebral bone mineral apparent density decreased during pregnancy and 6 months of lactation, followed by an increase during the 3 months after weaning. Calcium, phosphate, 25-hydroxyvitamin D, and osteocalcin increased significantly from midpregnancy to weaning whereas 1,25-dihydroxyvitamin D values showed significant decreases. Histomorphometric measurements from bone biopsies showed significant increases in most parameters of bone formation between pregnancy and weaning. These results are consistent with the hypothesis that at midpregnancy bone formation is decreased and cancellous bone resorption may have increased. During lactation, losses occur in both cortical and cancellous bone, partially depleting the maternal reservoir of calcium, but a subsequent increase in bone formation enables restoration of bone mineral after weaning to values similar to those in the control group.
A Trans-Acting Enhancer Modulates Estrogen-Mediated Transcription of Reporter Genes in Osteoblasts
Authors: Sasaki-Iwaoka Haruna , Maruyama Kazunori , Endoh Hideki , Komori Toshihisa , Kato Shigeaki , Kawashima Hiroyuki 
Published: April 2006

Abstract
The presence of bone-specific estrogen agonists and discovery of the osteoblast-specific transcription factor (TF), Cbfa1, together with the discovery of synergism between a TF Pit-1 and estrogen receptor α (ERα) on rat prolactin gene, led to investigation of Cbfa1 in the modulation of osteoblast-specific actions of estrogen. Reverse transcribed-polymerase chain reaction demonstrated expression of Cbfa1 in the osteoblastic cell lines, MG63, ROS17/2.8, and MC3T3E1, but not in nonosteoblastic cell lines, MCF7, C3H10T1/2, and HeLa. An ER expression vector and a series of luciferase (Luc) reporter plasmids harboring the Cbfa1 binding site OSE2 (the osteoblast-specific cis element in the osteocalcin promoter) and palindromic estrogen response elements (EREs) were cotransfected into both osteoblastic and nonosteoblastic cells. OSE2 worked as a cis- acting element in osteoblastic cells but not nonosteoblastic cells, whereas EREs were cis- acting in all cell lines. Synergistic transactivation was observed in osteoblastic cells only when both ERE and OSE2 were placed in juxtaposition to the promoter. Forced expression of Cbfa1 in C3H10T1/2 cells also induced synergism. Tamoxifen, a partial agonist/antagonist of estrogen, acted as an osteoblast-specific agonist in cells transfected with a promoter containing ERE and acted synergistically with a promoter containing the ERE-OSE2 enhancer combination. These results support the idea that bone-specific TFs modulate the actions of estrogen in a tissue-specific manner.
Marked Osteoblastopenia and Reduced Bone Formation in a Model of Multiple Myeloma Bone Disease in Severe Combined Immunodeficiency Mice
Authors: Hjorth-Hansen Henrik , Seifert Mark F. , Börset Magne , Aarset Harald , Östlie Agnes , Sundan Anders , Waage Anders 
Published: April 2006

Abstract
We report on an in vivo model of human myeloma producing bone disease in irradiated severe combined immunodeficiency disease mice using the human myeloma cell line JJN-3 and its subline JJN-3 T1. The cell lines are not Epstein-Barr virus transformed and produce large amounts of hepatocyte growth factor (HGF). Mice had radiological signs of osteolysis and mild hypercalcemia. Xenografted cells were predominantly found in bone marrow and brown adipose tissue, but also in meninges and liver. Take was documented by histopathological examination, immunophenotyping of cultured bone marrow, and radiography. HGF was detected in serum and bone marrow plasma. Disease generally occurred within 45 days of intravenous inoculation and was signaled by paraparesis or signs of intracranial neoplasia. More than 90% of the mice had take of xenografts. The subline JJN-3 T1 gave more reproducible bone marrow take than the native cell line. Bone histomorphometric examination revealed a 99% reduction in osteoblast counts and a 33% reduction in osteoclast counts in areas of tumor growth. Bone formation rates were reduced by 53%. The results suggest that osteoblastopenia and reduced bone formation is of importance for the occurrence of osteolytic lesions in this model.
The Primary Calcification in Bones Follows Removal of Decorin and Fusion of Collagen Fibrils
Authors: Hoshi Kazuto , Kemmotsu Shinichi , Takeuchi Yasuhiro , Amizuka Norio , Ozawa Hidehiro 
Published: April 2006

Abstract
To elucidate the mechanisms of primary calcification in bone, ultrastructural changes in collagen fibrils, as well as cytochemical alteration of proteoglycan, especially decorin, were investigated morphologically in 19-day postcoitum embryonic rat calvariae. Below the osteoblast layer, calcification of the osteoid area increased in direct proportion to its distance from the osteoblasts. In the uncalcified osteoid area, collagen fibrils near matrix vesicles possessed sharp contours and were a uniform 50 nm in diameter. Immunoelectron microscopy revealed decorin to be abundantly localized in the vicinity of the collagen fibrils. In the osteoid area undergoing the process of calcification, collagen fibrils tended to fuse side by side. Where calcification was progressed, this fusion was even more so. Some very large fibrils exhibited complicated contours, 400 nm or more in diameter. Although the calcification at this stage affected areas both inside and outside of the collagen fibrils, the interior areas manifested a lower density of calcification. The immunolocalization of decorin was also much decreased around these fibrils. Thus, primary calcification in bone matrix follows the removal of decorin and fusion of collagen fibrils. This phenomenon may aid in the process of calcification and bone formation, because (1) inhibitors of calcification, such as decorin, are removed, (2) the fusion of collagen fibrils provides the room necessary for rapid growth of mineral crystals, and (3) the soft elastic bone matrix containing abundant fused collagen fibrils less subjective to calcification is safe for both maternal and embryonic bodies and is convenient for subsequent bone remodeling.
Emerging Insights into the Role of Calcium Ions in Osteoclast Regulation
Authors: Zaidi Mone , Adebanjo Olugbenga A. , Moonga Baljit S. , Sun Li , Huang Christopher L.-H. 
Published: April 2006

Abstract
Osteoclasts are exposed to unusually high, millimolar, Ca2+ concentrations and can “sense” changes in their ambient Ca2+ concentration during resorption. This results in a sharp cystolic Ca2+ increase through both Ca2+ release and Ca2+ influx. The rise in cystolic Ca2+ is transduced finally into an inhibition of bone resorption. We have shown that a type 2 ryanodine receptor isoform, expressed uniquely in the osteoblast plasma membrane, functions as a Ca2+ influx channel, and possibly as a Ca2+ sensor. Ryanodine receptors are ordinarily microsomal membrane Ca2+ release channels. They have only recently been shown to be expressed a other sites, including nuclear membranes. At the latter site, ryanodine receptors gate nucleoplasmic Ca2+ influx. Nucleoplasmic Ca2+, in turn, regulates key nuclear processes, including gene expression and apoptosis. Here, we review potential mechanisms underlying the recognition, movement, and actions of Ca2+ in the osteoclast.
Growth Hormone Increases Cortical and Cancellous Bone Mass in Young Growing Rats with Glucocorticoid-Induced Osteopenia
Authors: Dr. Ørtoft Gitte , Andreassen Troels T. , Oxlund Hans 
Published: April 2006

Abstract
The effects of growth hormone (GH) on linear growth, bone formation, and bone mass have been examined in glucocorticoid (GC)-injected young growing rats. Two-month-old female Wistar rats were injected for 90 days with 1, 3, 6, or 9 mg of methylprednisolone alone or in combination with 5 mg of GH. Bone mass and bone formation parameters were examined in the femoral cortical bone and in cortical bone and cancellous bone of the lumbar vertebra. GC administration dose dependently decreased growth, longitudinal growth of the vertebra, as well as the modeling drift of the cortical bone of the vertebral body and femoral diaphysis. In the vertebral cancellous bone, GC also decreased the mineralizing surface and inhibited the growth-related increase in cancellous bone volume. GH increased growth, longitudinal growth of the vertebra, as well as the modeling drift of the vertebral body and the femoral diaphysis, resulting in an increased cortical bone mass. GH also increased cancellous bone volume and the mineralizing surface of the vertebral body. In GC-injected animals, GH normalized and further increased growth, longitudinal growth, and the modeling drift of both the femoral diaphysis and the vertebral body, resulting in an increased cortical bone mass at both locations. GH also increased cancellous bone volume of the vertebral body in GC-injected animals, but GH did not, however, reverse the decreased mineralizing surface of cancellous bone induced by GC injections. In conclusion, GC administration to growing rats retards normal growth, longitudinal growth, and cortical bone modeling drift. It also decreases the cancellous bone mineralizing surface and inhibits the normal age-related increase in cancellous bone volume of the vertebral body. In the growing rat skeleton, GH can counteract these GC-induced side effects, except for the GC-induced decrease in the mineralizing surface of cancellous bone of the vertebral body, which remained unaffected by GH administration.
The Effects of Gonadectomy on Bone Size, Mass, and Volumetric Density in Growing Rats Are Gender-, Site-, and Growth Hormone–Specific
Authors: Zhang X. Z. , Kalu D. N. , Erbas B. , Hopper J. L. , Seeman E. 
Published: April 2006

Abstract
Peak volumetric bone mineral density (BMD) is determined by the growth in bone size relative to the mineral accrued within its periosteal envelope. Thus, reduced peak volumetric BMD may be the result of reduced mineral accrual relative to growth in bone size. Because sex steroids and growth hormone (GH) influence bone size and mass we asked: What are the effects of gonadectomy (Gx) on bone size, bone mineral content (BMC), areal and volumetric BMD in growing male and female rats? Does GH deficiency (GH−) reduce the amount of bone in the (smaller) bone, i.e., reduce volumetric BMD? Does GH– alter the effect of Gx on bone size and mineral accrual? Gx or sham surgery was performed at 6 weeks in GH− and GH replete (GH+) Fisher 344 male and female rats. Changes in bone size, volume, BMC, areal and volumetric BMD, measured using dual X-ray absorptiometry (DPX-L), were expressed as percentage of controls at 8 months (mean ± SEM). All results shown were significant (p &lt; 0.05 level) unless otherwise stated. In GH+ and GH− males, respectively, Gx was associated with: lower femur volume (24%, 25%), BMC (43%, 45%), areal BMD (21%, 14%), and volumetric BMD (30%, 28%); lower spine (L1–L3) volume (26%, 28%), BMC (26%, 30%), and areal BMD (28%, 12%), but not volumetric BMD. Following Gx, GH+ females had increased femur volume (11%), no effect on BMC, decreased areal BMD (6%) and decreased volumetric BMD (17%); GH− females had no change in femur volume, but decreased femur BMC (24%), areal BMD (10%), and volumetric BMD (25%). In GH+ and GH− females, respectively, Gx was associated with a decrease in spine (L1–L3) BMC (12%, 15%), areal BMD (16%, 15%), and volumetric BMD (10%, 16%) with no change in volume. Deficits in non-Gx GH− relative to non-Gx GH+ (males, females, respectively) were: femur BMC (49%, 37%), areal BMD (23%, 8%), volume (19%, 19%) and volumetric BMD (37%, 22%); spine (L1–L3) BMC (46%, 42%), areal BMD (37%, 43%), volume (10%, 15%), and volumetric BMD (40%, 33%). Testosterone and GH are growth promoting in growing male rats, producing independent effects on bone size and mass; deficiency produced smaller appendicular bones with reduced volumetric BMD because deficits in mass were greater than deficits in size. At the spine, the reduction in size and accrual were proportional, resulting in a smaller bone with normal volumetric BMD. In growing female rats, estrogen was growth limiting at appendicular sites; deficiency resulted in a GH-dependent increase in appendicular size, relatively reduced accrual, and so, reduced volumetric BMD in a bigger bone. At the spine, accrual was reduced while growth in size was normal, thus volumetric BMD was reduced in the normal sized bone. Understanding the pathogenesis of low volumetric BMD requires the study of the differing relative growth in size and mass of the axial and appendicular skeleton in the male and female and the regulators of the growth of these traits.
Bone Mineralization and Turnover in Preterm Infants at 8–12 Years of Age: The Effect of Early Diet
Authors: Dr. Fewtrell M. S. , Prentice A. , Jones S. C. , Bishop N. J. , Stirling D. , Buffenstein R. , Lunt M. , Cole T. J. , Lucas A. 
Published: April 2006

Abstract
Our previous studies raised two hypotheses: first that suboptimal early nutrition and second that human milk have enhancing effects on long-term bone mineralization. To test these hypotheses experimentally, we measured whole body and regional bone mineral content (BMC) and bone mineral density (BMD), using dual-energy X-ray absorptiometry and single-photon absorptiometry, and bone turnover at 8–12 years in 244 preterm children (128 boys) who participated in a prospective randomized study of diet during the neonatal period. Dietary randomizations studied were: banked human milk (BBM, n = 87) versus preterm formula (PTF, n = 96) as the sole diet or as a supplement to mother's expressed breast milk (EBM); PTF (n = 25) versus term formula (TF, n = 36) as sole diet. Ninety-five term children of the same age were also studied. First, preterm children were shorter and lighter than term children (height SD scores −0.49 (1.1) vs. +0.22 (0.9), weight SD scores −0.41 (1.2) vs. +0.38 (1.0) and had significantly lower whole-body BMC than their peers; decrements were also evident at some regional sites. These differences disappeared after adjusting for bone area, body size, and pubertal status. Second, children previously randomized to BBM versus PTF or TF versus PTF showed no significant differences in anthropometry, BMC, BMD, or osteocalcin (OC). Third, there was no independent effect of the proportion of EBM on BMC, BMD, or OC and no interaction between randomized diet and the amount of EBM received. Fourth, plasma OC was significantly higher in preterm children than in term children (12.4 vs. 11.0 ng/ml, p &lt; 0.005) and in preterm children who had received a low-nutrient (BBM/TF) as opposed to a high-nutrient diet (PTF) during the neonatal period (12.9 vs. 11.9 ng/ml, p = 0.03). In conclusion, preterm children are shorter, lighter, and have lower bone mass than their peers at age 8–12 years. The lower BMC is, however, appropriate for the bone and body size achieved. Despite large differences in early mineral intake, early diet does not affect bone mass in preterm children, and fresh human milk has no specific effect. However, poor nutrition during the neonatal period may result in higher bone formation rates during childhood.