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Bilezikian, J.P.1
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Rodan, G.A.3
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1842377238
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Giant cell tumors of bone (osteoclastomas) are rare bone tumors, but they provide a source of mature human osteoclasts that are otherwise impossible to extract from the hard matrix of bone. These cells are not the neoplasic cell type and have a normal phenotype and function [M. Horton et al., J. Pathol. 144, 282 (1984)].
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1842329500
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note
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2, were seeded with the cell suspension for 2 hours. Nonadherent cells were rinsed off. The dentine slices, including an enriched population of osteoclasts, were cultured for 24 hours and fixed for 10 min in a 50:50 mixture of MEM with fixation buffer (3.5% paraformaldehyde and 2% sucrose in phosphate-buffered saline (PBS)].
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8
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1842334828
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note
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2, thickness 100 μm), polished on 3MM paper, washed in water to remove debris, sterilized in 70% ethanol for 5 s, and dried. Slices were biotinylated by first washing them in 50 mM sodium bicarbonate buffer, pH 8.6, for 1 hour and then adding 500 μl of biotinylation buffer per dentine slice [490 μl of sodium bicarbonate buffer with 10 μl of biotinylatton reagent, biotinamidocaproate N-hydroxysuccinamide ester (Amersham, UK)]. Biotinylation of matrix proteins proceeded for 1 hour at room temperature with mixing, followed by extensive washing in PBS for 3 hours.
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9
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0027166680
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2, 0.5% Triton X-100 (Pierce and Warner, Chester, UK), and 0.5% sodium azide, in PBS at pH 7.0]. Biotinylated matrix proteins were probed with streptavidinfluorescein (10 μg/ml, Vector Laboratories, UK). Rabbit polyclonal antibodies to native type I collagen were used at a 1/50 dilution (AB749, Chemicon, UK) with fluorescein isothiocyanate (FITC)-conjugated swine antibody to rabbit at a 1/40 dilution (Dakopatts, Denmark). Mouse monoclonal antibody (MAB A7), specific for degraded type I collagen, was provided by M. Bonde. Antibody MAB A7 was used at 10 μg/ml with FITC-conjugated goat antibody to mouse at a 1/20 dilution (Dakopatts, Denmark). Confocal microscopy was done as reported [P. Lakkakorpi, M. H. Helfrich, M. A. Horton, H. K. Väänänen, J. Cell Sci. 104, 663 (1993)] using the Leica TCS NT and Bio-Rad MRC 600 or 1020 systems. No specific staining was seen if primary antibodies were omitted (13). Photomultiplier tube voltage thresholds for confocal microscopy were set to gate out background fluorescence given by immunoglobulin G (IgG)-negative controls. Fluorescent images were sequentially collected in 1-μm steps through osteoclasts for FITC and tetramethylrhodamine isothiocyanate fluorochromes at 488-and 568-nm emission wavelengths, respectively. The images were analyzed with Voxel View (Vital Images, Fairfield, IA) and Iris Indigo Graphics (Silicon Graphics, Mountain View, CA).
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Lakkakorpi, P.1
Helfrich, M.H.2
Horton, M.A.3
Väänänen, H.K.4
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1842405119
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note
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Biotinylation provided a suitable label for matrix proteins including type I collagen as assessed by SDS-polyacrylamide gel electrophoresis (PAGE). Biotinylation did not affect resorption; cell attachment and the number and area of resorption pits were comparable with resorption cultures on unlabeled dentine substrates (73).
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12
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0347593587
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Inhibition of bone resorption with the cysteine proteinase inhibitor E64 (0.2 mM) (3) abolished the uptake of dentine matrix proteins (13).
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17
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1842368831
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Type I collagen was detected in 3% of osteoclasts in human fetal (14-week gestational age) bone sections. The lower frequency compared with osteoclast cultures reflects both the lower rate of resorption seen in vivo and the probability that many osteoclasts were not actively resorbing, even if on bone surfaces.
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Antibody MAB-A7 was raised to an eight-amino acid sequence in the COOH-terminal telopeptide region of type I collagen, which is concealed in its native form and is exposed on degradation [M. Bonde, P. Quist, C. Fledelius, B. J. Riis, C. Christiansen, Clin. Chem. 40, 2022 (1994)].
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K. Simmons and S. Fuller, Annu. Rev. Cell Biol. 1, 243 (1985); J. E. Rothman and F. T. Wieland, Science 272, 227 (1996); K. E. Mostov et al., Cold Spring Harbor Symp. Quant. Biol. 40, 775 (1995).
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Mostov, K.E.1
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1842328335
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note
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This research was supported by the Imperial Cancer Research Fund and The Wellcome Trust. We thank T. Chaplin, A. Edwards, M. Helfrich, and P. Jordan for their kind assistance and the orthopedic surgeons at Royal Orthopaedic Hosptial, Birmingham, UK, for supplying tumor samples.
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