bluuebunny/crossref_metadata_2025_split huggingface dataset:
Do More Highly Organized Collagen Fibrils Increase Bone Mechanical Strength in Loss of Mineral Density After One-Year Running Training?
Authors: Puustjärvi Kaija , Nieminen Jyrki , Räsänen Tuomas , Hyttinen Mika , Helminen Heikki J. , Kröger Heikki , Huuskonen Jouni , Alhava Esko , Kovanen Vuokko 
Published: April 2006

Abstract
The aim of this study was to evaluate the effects of long-term running training on the structural properties of bone. Ten beagle dogs ran according to a strenuous progressive program (up to 40 km/day) for 1 year. At the end of the training program, there was a significant reduction in bone mineral density (up to 9.7%) in the vertebrae of the runner dogs as compared with 10 sedentary control dogs. Polarized light microscopy of the vertebral trabecular bone, however, displayed proportionally higher retardation values of the collagen network of the runner dogs than of the sedentary dogs, suggesting a reorganization in a more parallel manner in the collagen fibrils. The concentration and cross-linking of collagen in the bones remained similar in both groups. No differences were observed in the force to failure of bones of the two groups nor in the histomorphometric analysis of the bones. We suggest that the collagen network in the bones accounted for the maintenance of the strength properties in the bones of the runner dogs despite the loss of mineral density.
In Vitro Responses to 17β-Estradiol Throughout Pubertal Maturation in Female Human Bone Cells
Authors: Dr. Delaveyne-Bitbol Roselyne , Garabedian Michèle 
Published: April 2006

Abstract
To test the hypothesis that bone sensitivity to estrogens differ with the pubertal status, we cultured human osteoblasts (hOBs) from 14 girls (3–18 years) and examined the effects of repeated weekly doses of 17β-estradiol (E2, 10 pM–10 nM) on estradiol receptor (ER) and progesterone receptor (PR) expression, type I procollagen (PICP) and osteocalcin (BGP; bone Gla protein) production, and alkaline phosphatase (ALP) activity. The bone samples were divided into two equal groups according to the pubertal status and plasma E2 level of the donor. The two groups were significantly different for age (9 ± 1 and 15 ± 1 years), pubertal status (Tanner stages I–III and IV–V), and plasma E2 concentrations (17 ± 3 and 49 ± 4 pg/ml). ER and PR were expressed and not influenced by the sexual maturation in untreated cells. E2 increased ER in the two groups with nanomolar doses. Picomolar doses did not significantly increase ER expression but led to significant differences in the percentage of cells expressing ER in premenarchial (33%) and postmenarchial (7%) hOB cultures. In the two groups, E2 had no clear effect on PR expression, ALP activity, nor BGP production. But repeated weekly doses of E2 significantly influenced PICP production at picomolar doses. This effect depended upon the sexual maturation of the donor. E2 decreased PICP in premenarchial cultures and increased PICP in postmenarchial cultures. Thus, E2 modulates in vitro human bone cell metabolism and probably their phenotype and has different effects, depending on the pubertal status of the donor. Unlike what could have been expected, prepubertal and early pubertal hOBs appear to be specifically sensitive to picomolar doses of E2, suggesting that this hormone is a crucial regulator of bone metabolism even before puberty.
Tartrate-Resistant Bone Acid Phosphatase: Large-Scale Production and Purification of the Recombinant Enzyme, Characterization, and Crystallization
Authors: Kaija Helena , Jia Jia , Lindqvist Ylva , Andersson Göran , Prof. Vihko Pirkko 
Published: April 2006

Abstract
Tartrate-resistant acid phosphatase (TRAP) is an enzyme expressed in bone-resorbing osteoclasts and certain tissue macrophages in human tissues. The functions of TRAP in biological systems are not known. Elucidation of the three-dimensional structure of the active site could yield important information about the physiological substrate(s) of the enzyme. We have produced recombinant rat bone TRAP using a baculovirus expression vector system. The production was scaled up to a 30-l bioreactor, and a method of purification in large scale was developed. The enzyme is composed of one 34 kDa polypeptide chain. Trypsin digestion resulted in a preparation where two subunits of ∼23 kDa and ∼16 kDa appeared after disulfide reduction. Trypsin digestion activated the enzyme. We generated monoclonal antibodies against recombinant TRAP. One of the selected antibodies detected the 23 kDa subunit in Western blotting. The reduced and oxidized forms of the enzyme could be separated by Mono-S cation-exchange chromatography. Crystals of TRAP have been obtained with ammonium sulfate/polyethylene glycol as precipitant. They belong to space group P212121 or P21212 with unit cell dimensions a = 57.2 Å, b = 69.5 Å, and c = 87.2 Å and diffract to at least 2.2 Å resolution. A packing density value of 2.55 Å3/Da is consistent with one subunit in the asymmetric unit.
Demonstration of the Predominant Urine Osteocalcin Fragments Detectable by Two-Site Immunoassays
Authors: Matikainen Terhi , Käkönen Sanna-Maria , Pettersson Kim , Karp Matti , Lövgren Timo , Väänänen H. Kalervo , Hellman Jukka 
Published: April 2006

Abstract
We have isolated and characterized human osteocalcin (OC) fragments from pubertal urine. The fragments were isolated by immunoaffinity chromatography based on monoclonal antibody 6F9 and further purified by reverse phase chromatography. The major isolated forms, which were detectable with two-site immunofluorometric assays for serum OC, span residues 6–30 and 7–30 as determined by mass spectrometry and N-terminal amino acid sequencing. Full-length OC was not detectable in the supernatant fraction of urine but could be extracted with guanidinium hydrochloride from the sediment of urine samples. Urine samples from subjects with different menopausal status were measured by two different two-site assays. Urine OC (uOC) concentrations were 12- to 16-fold higher in the pubertal group than in the adult group. Also, the uOC concentration in a postmenopausal group was significantly higher than in a premenopausal group. The difference was 125% and 75% (values for p &lt; 0.0001), respectively, when measured with the two assays. uOC concentrations in postmenopausal subjects on hormone replacement therapy were indistinguishable from the premenopausal subjects. The fact that uOC can be measured by a noncompetetive two-site assay design offers improved analytical sensitivity. Urine as the sample matrix is also especially interesting because the predominant markers of bone resorption, collagen type I peptides or cross-links, are performed on urine samples. Our results from the technical validation of two-site assays for uOC and from applying these to human pubertal and pre- and postmenopausal samples calls for more extensive clinical validation.
Parathyroid Hormone and Mechanical Usage Have a Synergistic Effect in Rat Tibial Diaphyseal Cortical Bone
Authors: Ma Yanfei , Jee Webster S. S. , Yuan Zhongzhi , Wei Wei , Chen Hongka , Pun Sunwah , Liang Haohai , Lin Chaohua 
Published: April 2006

Abstract
Previous reports showed that bone mass and architecture only partially recovered by remobilization (RM) after immobilization (IM)-induced osteopenia, and that parathyroid hormone (PTH) had an anabolic effect on the skeleton. The aim of this study was to determine whether low doses of PTH could restore IM-induced cortical bone loss and whether a combination of PTH plus loading (RM) treatment would be more effective than the PTH in unloaded (IM) limbs. One hundred and sixty 6-month-old rats were divided into aging and IM groups. The right hindlimb of the rat was immobilized by elastic bandage for 18 weeks, and then groups of rats were either kept IM or RM and treated with 30 μg or 80 μg of hPTH(1–38)/kg/day for 2, 10, and 20 weeks. Fluorescent-labeled, undecalcified cross-sections of right tibial shafts were studied. We found that RM for 20 weeks after 18 weeks of IM only partially recovered IM-induced muscle weight loss and PTH had no effect on muscle weight in either IM or RM limbs; that RM for 20 weeks after 18 weeks of IM partially restored some minimal cortical width by stimulating periosteal and endocortical bone formation and decreasing endocortical resorption; that PTH treatment of IM limbs completely restored IM-induced cortical bone loss and added extra bone by stimulating bone formation indices on all bone surfaces and depressing bone resorption on endocortical surface; that PTH treatment of RM limbs produced similar anabolic effects as in IM limbs with 30 μg/kg/day dose but the 80 μg/kg/day dose-treated limbs had a higher periosteal bone formation rate, which created a larger cross-sectional area, more cortical bone area, and a thicker cortex than the same dose treated IM limbs; and that PTH 80 μg/kg/day treatment produced more anabolic effect than the 30 μg/kg/day in both IM and RM limbs. We concluded that reloading the hindlimb by RM after long-term IM could not recover the cortical bone mass. PTH at employed doses was able to completely restore IM-induced cortical bone loss, and this effect was independent of mechanical stimulation. However, when PTH was combined with mechanical loading (RM), a synergistic anabolic effect on periosteal bone formation occurred which increased the cross sectional area that can increase bone strength.
Extension of Phenotype Associated with Structural Mutations in Type I Collagen: Siblings with Juvenile Osteoporosis Have an α2(I)Gly436 → Arg Substitution
Authors: Dawson Paul A. , Kelly Thaddeus E. , Marini Joan C. 
Published: April 2006

Abstract
Mutations in the type I collagen genes have been identified as the cause of all four types of osteogenesis imperfecta (OI). We now report a mutation that extends the phenotype associated with structural abnormalities in type I collagen. Two siblings presented with a history of back pain and were diagnosed with juvenile osteoporosis, based on clinical and radiological examination. Radiographs showed decreased lumbar bone density and multiple compression fractures throughout the thoracic and lumbar spines of both patients. One child has moderate short stature and mild neurosensory hearing loss. However, neither child has incurred the long bone fractures characteristic of OI. Protein studies demonstrated electrophoretically abnormal type I collagen in samples from both children. Enzymatic cleavage of RNA:RNA hybrids identified a mismatch in type I collagen α2 (COL1A2) mRNA. DNA sequencing of COL1A2 cDNA subclones defined the mismatch as a single-base mutation (1715G → A) in both children. This mutation predicts the substitution of arginine for glycine at position 436 (G436R) in the helical domain of the α2(I) chain. Analysis of genomic DNA identified the mutation in the asymptomatic father, who is presumably a germ-line mosaic carrier. The presence of the same heterozygous mutation in two siblings strongly suggests that the probands display the full phenotype. Taken together, the clinical, biochemical, and molecular findings of this study extend the phenotype associated with type I collagen mutations to cases with only spine manifestations and variable short stature into adolescence.
BMP-6 Is an Autocrine Stimulator of Chondrocyte Differentiation
Authors: Grimsrud Christopher D. , Romano Paul R. , D'souza Mary , Puzas J. Edward , Reynolds Paul R. , Rosier Randy N. , O'keefe Regis J. 
Published: April 2006

Abstract
While parathyroid hormone-related protein (PTHrP) has been characterized as an important negative regulator of chondrocyte maturation in the growth plate, the autocrine or paracrine factors that stimulate chondrocyte maturation are not well characterized. Cephalic sternal chondrocytes were isolated from 13-day embryos, and the role of bone morphogenetic protein-6 (BMP-6) as a positive regulator of chondrocyte maturation was examined in monolayer cultures. Progressive maturation, which was accelerated in the presence of ascorbate, occurred in the cultures. During maturation, the cultures expressed high levels of BMP-6 mRNA which preceded the induction of type X collagen mRNA. Treatment of the cultures with PTHrP (10−7 M) at the time of plating completely abolished BMP-6 and type X collagen mRNA expression. Removal of PTHrP after 6 days was followed by the rapid (within 24 h) expression of BMP-6 and type X collagen mRNA, with BMP-6 again preceding type X collagen expression. The addition of exogenous BMP-6 (100 ng/ml) to the cultures accelerated the maturation process both in the presence and absence of ascorbate and resulted in the highest levels of type X collagen. When exogenous BMP-6 was added to PTHrP containing cultures, maturation occurred with the expression of high levels of type X collagen, despite the presence of PTHrP in the cultures. Furthermore, BMP-6 did not stimulate expression of its own mRNA in the PTHrP treated cultures, but it did stimulate the expression of Indian hedgehog (Ihh) mRNA. These latter findings suggest that while PTHrP directly inhibits BMP-6, it indirectly regulates Ihh expression through BMP-6. Other phenotypic changes associated with chondrocyte differentiation were also stimulated by BMP-6, including increased alkaline phosphatase activity and decreased proliferation. The results suggest that BMP-6 is an autocrine factor that initiates chondrocyte maturation and that PTHrP may prevent maturation by inhibiting the expression of BMP-6.
Characterization of Mouse Cathepsin K Gene, the Gene Promoter, and the Gene Expression
Authors: Li Yi-Ping , Chen Wei 
Published: April 2006

Abstract
Cathepsin K, a lysosomal cysteine protease, is abundantly and selectively expressed in osteoclasts and has a specialized role in osteoclast-mediated bone resorption. In contrast to function studies, transcription regulation of cathepsin K remains largely unknown. In this study, the gene encoding mouse cathepsin K and the promoter have been isolated and completely sequenced. In addition, the temporal and spatial expressions of cathepsin K have been characterized. Intrachromosomal mapping studies revealed that the gene contains eight exons and seven introns spanning ∼10.6 kb of genomic DNA, a genomic organization that was highly conserved with respect to its human homology. Analysis of the 9 kb 5′ flanking region indicates that this gene lacks canonical TATA and CAAT boxes and contains multiple putative transcription regulatory elements which are also present in the comparable position of 5′ flanking region of human cathepsin K gene. Mouse cathepsin K was found to be a single-copy gene. Northern blot analysis of RNAs from a number of mouse tissues revealed that cathepsin K mRNA is selectively expressed in osteoclast. The selective expression of cathepsin K was confirmed by anticathepsin K immunohistochemical staining. The sequence of cathepsin K expression was linked to osteoclast differentiation in vivo and in vitro by a tartrate-resistant acid phosphatase–anticathepsin K dual immunostaining technique. Cathepsin K is initially expressed at the preosteoclast stage and throughout the mature osteoclast stage. The primer extension assay indicated a major transcription start site 58 bp upstream of the initiator Met codon. The characterization of the cathepsin K gene, its promoter, and the temporal and spatial expression may provide valuable insights into its osteoclast-specific expression and the molecular mechanisms responsible for osteoclast activation.
Differential Effects of 20-Epi Vitamin D Analogs on the Vitamin D Receptor Homodimer
Authors: Dr. Koszewski Nicholas J. , Reinhardt Timothy A. , Horst Ronald L. 
Published: April 2006

Abstract
Vitamin D analogs have received increased attention because of their possible therapeutic benefits in treating osteoporosis and various proliferative disorders. Several analogs were examined for their effects on DNA binding of the vitamin D receptor (VDR) homodimer complex with the murine osteopontin vitamin D response element. All of the tested analogs increased complex binding by recombinant human VDR in the electrophoretic mobility shift assay and notable differences in mobility of these complexes were observed. A panel of C-terminal anti-VDR antisera were screened for their ability to interact with analog-bound VDR homodimer complexes or as a heterodimer complex with recombinant human retinoid X receptor α (rhRXRα). Like calcitriol, analog-bound heterodimer complexes were largely resistant to interaction with these antisera; however, striking differences were observed with the various antisera in an analogous homodimer binding experiment. KH1060 and CB1093, analogs with 20-epi conformations, produced homodimer complexes that were 3- to 6-fold more resistant to supershifting with Ab180 compared with the hormone or EB1089. Chymotrypsin digestion in combination with Western blotting using a C-terminal anti-VDR antiserum revealed similar digestion patterns for all ligands. However, KH1060- and CB1093-bound VDR complexes were more resistant to digestion than either calcitriol or EB1089. Finally, the ability of these compounds to yield stable homodimer complexes was assessed by challenging preformed homodimer with the exogenous addition of rhRXRα extracts. Although new heterodimer complexes appeared in a time-dependent fashion, the preformed homodimer complexes exhibited stable binding throughout the time course of the experiment. The results indicate that VDR homodimers are targets of vitamin D analogs with differential effects on C-terminal protein conformation that may partially explain the varied biological responses of these compounds.
Osteogenic Phenotype Expression of Allogeneic Rat Marrow Cells in Porous Hydroxyapatite Ceramics
Authors: Akahane M. , Ohgushi H. , Yoshikawa T. , Sempuku T. , Tamai S. , Tabata S. , Dohi Y. 
Published: April 2006

Abstract
Porous hydroxyapatite (HA) ceramics were combined with either allogeneic (ACI) or isogeneic (Fischer 344) rat marrow cells and implanted in subcutaneous sites of Fischer rats. FK506 as an immunosuppressant or saline was administered to the recipient rats. The implanted marrow/HA composites were harvested on day 28 and analyzed for bone-forming capability by determining osteoblastic phenotype expression levels of protein synthesis and gene expression. The alkaline phosphatase (ALP) activity and osteocalcin (OC) contents were very low and mRNAs (Northern blot analysis) were not detected in the allografts without FK506. However, high activity of ALP and high content of OC were found and mRNAs were detected in the allografts with FK506 and in the isografts (with and without FK506). This analysis indicates the osteogenic potential of allogeneic marrow cells in the presence of FK506. The histologic sections revealed that allografts without FK506 did not show bone formation but did show the infiltration of many small cells in the ceramics indicating an immunologic reaction, however, the allografts with FK506 and the isografts (with and without FK506) showed consistent de novo bone formation on the HA pore surface. These results indicate that FK506 can suppress the immunologic reaction in the allografts and induce a favorable conditions to support osteoblastic differentiation of allogeneic rat marrow stromal stem cells on the surface of HA ceramics. Therefore, our study suggests the feasibility of clinical transplantation of allogeneic bone marrow for a selected bone graft in applications using adjuvant systemic immunosuppression.
An Automated Analysis of Intracortical Porosity in Human Femoral Bone Across Age
Authors: Dr. Stein M. S. , Feik S. A. , Thomas C. D. L. , Clement J. G. , Wark J. D. 
Published: April 2006

Abstract
The matrix of human cortical bone is arranged around a network of vascular spaces (hereafter referred to as “pores”). Our aim was to investigate age-related differences in human cortical porosity (total pore area divided by cortical bone area), pore size and number, and surface to volume ratios, while adjusting for sex, height, and weight. Ninety-six specimens of entire transverse sections of human femoral diaphysis, from subjects aged 21–92 years, were examined. We used our established automated image acquisition and analysis system which measures pores from entire sections of multiple specimens of bone. Over 400,000 pores were recorded. Results showed a greater porosity in older bone (p &lt; 0.01) but marked variation in porosity for any given age. The cohort median, of the specimen medians, of pore cross-sectional area was 2050 μm2. Older specimens did not have more pores than younger specimens but had a greater proportion of larger pores (p &lt; 0.05) and greater intraspecimen variation in pore size (p &lt; 0.001). The pore surface to bone matrix volume ratio was a median 2.3 mm2/mm3. This varied more than 4-fold between individuals but did not relate to age. No simple relationships were found between any of the measured parameters and either sex, height, or weight, even after adjustment for age. We conclude that the greater porosity in older specimens is due to greater pore size rather than a larger number of pores. Age, however, explains little of the inter-individual variation in the parameters studied.
Bone Loss in Celiac Disease Is Related to Secondary Hyperparathyroidism
Authors: Dr. Selby Peter L. , Davies Michael , Adams Judith E. , Mawer E. Barbara 
Published: April 2006

Abstract
Celiac disease is a major cause of intestinal malabsorption. Previous studies have demonstrated that celiac disease is associated with significant osteoporotic bone loss. These studies have suggested that successful treatment of the malabsorption is associated with amelioration of the bone loss. Such studies have failed to examine bone mass at peripheral skeletal sites which is more likely to be responsive to changes in parathyroid hormone (PTH) in response to calcium malabsorption. We have examined bone density in the lumbar spine, femoral neck, and distal forearm in 35 patients with celiac disease who had been established on gluten-free diet. In addition, the concentrations of PTH and 1,25-dihydroxyvitamin D (1,25(OH)2D) were measured. Bone density was below that expected for the subject's age and gender at all sites. This was most marked in the distal forearm where the bone density was 1.40 SD below expected (p &lt; 0.0001). In the forearm, there was a negative relationship between bone density and PTH concentration (r = −0.49, p = 0.009). In the forearm and lumbar spine, there was a negative relationship between 1,25(OH)2D concentration and bone density. Bone mass was not related to the concentration of 25-hydroxyvitamin D at any of the skeletal sites measured. Bone density is reduced in the peripheral skeleton in celiac disease and this deficit persists despite treatment with apparent normalization at axial skeletal sites. This reduction in bone mass is related to the presence of secondary hyperparathyroidism which should be sought in all patients with treated celiac disease.
Expression and Localization of Estrogen Receptor-β in Murine and Human Bone
Authors: Vidal Olle , Kindblom Lars-Gunnar , Ohlsson Claes 
Published: April 2006

Abstract
Estrogens have profound effects on bone metabolism. Cellular responses to estrogens are mediated by estrogen receptors (ERs) which belong to the nuclear receptor superfamily. Two estrogen receptors, ERα and ERβ, have been cloned. Previously expression of ERα has been shown in osteoblasts. Here we demonstrate that the transcript for ERβ can be detected in the human osteosarcoma cell lines (MG-63 and SaOS-2) and in cultured human osteoblast-like cells. We also show that ERβ protein is present in nuclear extracts from these cells. Furthermore, ERβ immunoreactivity is found in sections of murine and human bone. Murine and human osteoblast and osteocyte nuclei are immunoreactive for ERβ. Osteoclasts are also ERβ immunoreactive but the staining is mainly cytoplasmic. The present study demonstrates that ERβ is present in all the cellular compartments involved in bone formation and bone resorption, both in human and in murine bone tissue.