Name: analysis of minerals produced by hfob 1.19 and saos-2 cells using transmission electron microscopy with energy dispersive x-ray microanalysis
Uploaded: 2018-06-24T13:00:00
Description: Watch this Scientific Journal Video about Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis at JoVE.com

MK 和要求执行手工操作和 LB 准备的图纸和制作的电影。问写的手稿, LB 写的剧本和 MK 准备的表。SM、RB 和 SP 认真阅读了表、剧本和手稿。作者感谢汉娜 Chomontowska 为 ultramicrotomy 以及叔父 Suski 和亨瑞 Bilski 提供了出色的协助, 他们对 TEM EDX 分析提供了出色的帮助。作者要感谢帕特里克林博士的专业英语语言更正和芭芭拉 Sobiak 记录的指示。
这项工作得到波兰科学和高等教育部赠款 N N401 140639 的支持, 由国家科学中心提供赠款, 波兰 2016/23/n/NZ4/03313 至磅和 2016/23/n/NZ1/02449 至 MK, 欧盟 FP7 项目 BIOIMAGINE: 生物成像在研究创新和教育, GA 264173, 和由 Nencki 实验生物学研究所的法定资金, 波兰科学院。

1. 细胞培养和治疗
<ol><li>将所有必要的材料放在层流罩下, 并在紫外线照射下消毒。文化媒介是: 1:1 混合火腿的 F12 和 DMEM 培养基与2.5 毫米 l-谷氨酰胺补充100的 u/毫升青霉素, 100 U/毫升链霉素, 0.3 毫克/毫升 G418 和10% 胎牛血清 (v/v) 为人类胎儿 hFOB 1.19 SV40 大 T 抗原转染成骨细胞, 和麦考伊的5A 培养基与1.5 毫米 l-谷氨酰胺补充 100 u/毫升青霉素, 100 u/毫升链霉素和15% 血清 (v/v) 的人骨肉瘤 Saos-2 细胞。</...

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	<li>Buckwalter, J. A., Cooper, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+structure+and+function.">Bone structure and function.</a> Instr. Course Lect. 36, 27-28 (1987).</li><li>Anderson, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+matrix+vesicles.">Molecular biology of matrix vesicles.</a> Clin. Orthop. Relat. Res. 314, 266-280 (1995).</li><li>Anderson, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+vesicles+and+calcification.">Matrix vesicles and calcification.</a> Curr Rheumatol. 5 (3), 222-226 (2003).</li><li>Bolean, M., Sim&#227;o, A. M. S., Barioni, M. B., Favarin, B. Z., Sebinelli, H. G., Veschi, E. A., Janku, T. A. B., Bottini, M., Hoylaerts, M. F., Itri, R., Mill&#225;n, J. L., Ciancaglini, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+aspects+of+biomineralization.">Biophysical aspects of biomineralization.</a> Biophys Rev. 9 (5), 747-760 (2017).</li><li>Bottini, M., Mebarek, S., Anderson, K. L., Strzelecka-Kiliszek, A., Bozycki, L., Sim&#227;o, A. M. S., Bolean, M., Ciancaglini, P., Bandorowicz Pikula, J., Pikula, S., Magne, D., Volkmann, N., Hanein, D., Mill&#225;n, J. L., Buchet, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+vesicles+from+chondrocytes+and+osteoblasts:+Their+biogenesis,+properties,+functions+and+biomimetic+models.">Matrix vesicles from chondrocytes and osteoblasts: Their biogenesis, properties, functions and biomimetic models.</a> Biochim Biophys Acta. 1862 (3), 532-546 (2018).</li><li>Hessle, L., Johnson, K. A., Anderson, H. C., Narisawa, S., Sali, A., Goding, J. W., Terkeltaub, R., Millan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-nonspecific+alkaline+phosphatase+and+plasma+cell+membrane+glycoprotein-1+are+central+antagonistic+regulators+of+bone+mineralization.">Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization.</a> Proc. Natl. Acad. Sci. U. S. A. 99 (14), 9445-9449 (2002).</li><li>Garimella, R., Bi, X., Anderson, H. C., Camacho, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+of+phosphate+substrate+as+a+major+determinant+of+mineral+type+formed+in+matrix+vesicle-mediated+in+vitro+mineralization:+An+FTIR+imaging+study.">Nature of phosphate substrate as a major determinant of mineral type formed in matrix vesicle-mediated in vitro mineralization: An FTIR imaging study.</a> Bone. 38 (6), 811-817 (2006).</li><li>Thouverey, C., Bechkoff, G., Pikula, S., Buchet, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inorganic+pyrophosphate+as+a+regulator+of+hydroxyapatite+or+calcium+pyrophosphate+dihydrate+mineral+deposition+by+matrix+vesicles.">Inorganic pyrophosphate as a regulator of hydroxyapatite or calcium pyrophosphate dihydrate mineral deposition by matrix vesicles.</a> Osteoarthr. Cartil. 17, 64-72 (2009).</li><li>Terkeltaub, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inorganic+pyrophosphate+generation+and+disposition+in+pathophysiology.">Inorganic pyrophosphate generation and disposition in pathophysiology.</a> Am. J. Phys. 281 (1), 1-11 (2001).</li><li>Guicheux, J., Palmer, G., Shukunami, C., Hiraki, Y., Bonjour, J. P., Caverzasio, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+in+vitro+culture+system+for+analysis+of+functional+role+of+phosphate+transport+in+endochondral+ossification.">A novel in vitro culture system for analysis of functional role of phosphate transport in endochondral ossification.</a> Bone. 27 (1), 69-74 (2000).</li><li>Yadav, M. C., Bottini, M., Cory, E., Bhattacharya, K., Kuss, P., Narisawa, S., Sah, R. L., Beck, L., Fadeel, B., Farquharson, C., Mill&#225;n, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+mineralization+deficits+and+impaired+biogenesis+and+function+of+chondrocyte-derived+matrix+vesicles+in+Phospho1(-/-)+and+Phospho1/Pi+t1+double-knockout+mice.">Skeletal mineralization deficits and impaired biogenesis and function of chondrocyte-derived matrix vesicles in Phospho1(-/-) and Phospho1/Pi t1 double-knockout mice.</a> J. Bone Miner. Res. 31 (6), 1275-1286 (2016).</li><li>Thouverey, C., Malinowska, A., Balcerzak, M., Strzelecka-Kiliszek, A., Buchet, R., Dadlez, M., Pikula, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+characterization+of+biogenesis+and+functions+of+matrix+vesicles+released+from+mineralizing+human+osteoblast-like+cells.">Proteomic characterization of biogenesis and functions of matrix vesicles released from mineralizing human osteoblast-like cells.</a> J. Proteome. 74 (7), 1123-1134 (2011).</li><li>Wang, W., Xu, J., Kirsch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin-mediated+Ca2++influx+regulates+growth+plate+chondrocyte+maturation+and+apoptosis.">Annexin-mediated Ca2+ influx regulates growth plate chondrocyte maturation and apoptosis.</a> J. Biol. Chem. 278 (6), 3762-3769 (2003).</li><li>Nollet, M., Santucci-Darmanin, S., Breuil, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+osteoblasts+is+involved+in+mineralization+and+bone+homeostasis.">Autophagy in osteoblasts is involved in mineralization and bone homeostasis.</a> Autophagy. 10 (11), 1965-1977 (2014).</li><li>Boonrungsiman, S., Gentleman, E., Carzaniga, R., Evans, N. D., McComb, D. W., Porter, A. E., Stevens, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+intracellular+calcium+phosphate+in+osteoblast-mediated+bone+apatite+formation.">The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation.</a> Proc. Natl. Acad. Sci. U. S. A. 109 (35), 14170-14175 (2012).</li><li>Genge, B. R., Wu, L. N., Wuthier, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+modeling+of+matrix+vesicle+nucleation:+synergistic+stimulation+of+mineral+formation+by+annexin+A5+and+phosphatidylserine.">In vitro modeling of matrix vesicle nucleation: synergistic stimulation of mineral formation by annexin A5 and phosphatidylserine.</a> J. Biol. Chem. 282 (36), 26035-26045 (2007).</li><li>Jahnen-Dechent, W., Sch&#228;fer, B., Ketteler, M., McKee, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mineral+chaperones:+a+role+for+fetuin-A+and+osteopontin+in+the+inhibition+and+regression+of+pathologic+calcification.">Mineral chaperones: a role for fetuin-A and osteopontin in the inhibition and regression of pathologic calcification.</a> J. Mol. Med. (Berl). 86 (4), 379-389 (2008).</li><li>Suchanek, W., Yoshimura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+and+properties+of+hydroxyapatite-based+biomaterials+for+use+as+hard+tissue+replacement+implants.">Processing and properties of hydroxyapatite-based biomaterials for use as hard tissue replacement implants.</a> J. Miner. Res. 13 (1), 94-117 (1998).</li><li>Pautke, C., Schieker, M., Tischer, T., Kolk, A., Neth, P., Mutschler, W., Milz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+osteosarcoma+cell+lines+MG-63,+Saos-2+and+U-2+OS+in+comparison+to+human+osteoblasts.">Characterization of osteosarcoma cell lines MG-63, Saos-2 and U-2 OS in comparison to human osteoblasts.</a> Anticancer Res. 24 (6), 3743-3748 (2004).</li><li>Yen, M. -. L., Chien, C. -. C., Chiu, I. -. M., Huang, H. -. I., Chen, Y. -. C., Hu, H. -. I., Yen, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+differentiation+and+characterization+of+the+human+fetal+osteoblastic+1.19+cell+line:+a+possible+in+vitro+model+of+human+mesenchymal+progenitors.">Multilineage differentiation and characterization of the human fetal osteoblastic 1.19 cell line: a possible in vitro model of human mesenchymal progenitors.</a> Stem Cells. 25 (1), 125-131 (2007).</li><li>Brittle, S. W., Foose, D. P., O'Neil, K. A., Sikon, J. M., Johnson, J. K., Stahler, A. C., Ryan, J. D., Higgins, S. R., Sizemore, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+raman-based+imaging+method+for+characterizing+the+molecular+adsorption+and+spatial+distribution+of+silver+nanoparticles+to+hydrated+mineral+surfaces.">A raman-based imaging method for characterizing the molecular adsorption and spatial distribution of silver nanoparticles to hydrated mineral surfaces.</a> Environ Sci Technol. , (2018).</li><li>Liu, N., Wang, Y., Ge, F., Liu, S., Xiao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonistic+effect+of+nano-ZnO+and+cetyltrimethyl+ammonium+chloride+on+the+growth+of+Chlorella+vulgaris:+Dissolution+and+accumulation+of+nano-ZnO.">Antagonistic effect of nano-ZnO and cetyltrimethyl ammonium chloride on the growth of Chlorella vulgaris: Dissolution and accumulation of nano-ZnO.</a> Chemosphere. 196, 566-574 (2018).</li><li>Tasbihi, M., Ko&#269;&#236;, K., Troppov&#225;, I., Edelmannov&#225;, M., Reli, M., &#268;apek, L., Schom&#228;cker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photocatalytic+reduction+of+carbon+dioxide+over+Cu/TiO2+photocatalysts.">Photocatalytic reduction of carbon dioxide over Cu/TiO2 photocatalysts.</a> Environ Sci Pollut Res Int. , (2017).</li><li>Chen, N. X., O'Neill, K. D., Chen, X., Moe, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin-Mediated+Matrix+Vesicle+Calcification+in+Vascular+Smooth+Muscle+Cells.">Annexin-Mediated Matrix Vesicle Calcification in Vascular Smooth Muscle Cells.</a> J. Bone Miner. Res. 23 (11), 1798-1805 (2008).</li><li>Strzelecka-Kiliszek, A., Bozycki, L., Mebarek, S., Buchet, R., Pikula, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+minerals+in+vesicles+produced+by+human+osteoblasts+hFOB+1.19+and+osteosarcoma+Saos-2+cells+stimulated+for+mineralization.">Characteristics of minerals in vesicles produced by human osteoblasts hFOB 1.19 and osteosarcoma Saos-2 cells stimulated for mineralization.</a> J. Inorg. Bioch. 171, 100-107 (2017).</li><li>Thouverey, C., Strzelecka-Kiliszek, A., Balcerzak, M., Buchet, R., Pikula, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+vesicles+originate+from+apical+membran&#281;+microvilli+of+mineralizing+osteoblast-like+Saos-2+cells.">Matrix vesicles originate from apical membran&#281; microvilli of mineralizing osteoblast-like Saos-2 cells.</a> J. Cell. Biochem. 106 (1), 127-138 (2009).</li><li>Cazalbou, S., Eichert, D., Ranz, X., Drouet, C., Combes, C., Harmand, M. F., Rey, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+exchanges+in+apatites+for+biomedical+application.">Ion exchanges in apatites for biomedical application.</a> J. Mater. Sci. Mater. Med. 16 (5), 405-409 (2005).</li><li>Kraus, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consolidated+data+analysis+and+presentation+using+an+open-source+add-in+for+the+Microsoft+Excel&#174;+spreadsheet+software.">Consolidated data analysis and presentation using an open-source add-in for the Microsoft Excel&#174; spreadsheet software.</a> Med. Writ. 23 (1), 25-28 (2014).</li><li>Kawasaki, K., Buchanan, A. V., Weiss, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomineralization+in+humans:+making+the+hard+choices+in+life.">Biomineralization in humans: making the hard choices in life.</a> Annu. Rev. Genet. 43, 119-142 (2009).</li><li>Bonucci, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+mineralization.">Bone mineralization.</a> Front. Biosci. 17, 100-128 (2012).</li><li>Veis, A., Dorvee, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomineralization+mechanisms:+A+new+paradigm+for+crystal+nucleation+in+organic+matrices.">Biomineralization mechanisms: A new paradigm for crystal nucleation in organic matrices.</a> Calcif. Tissue Int. 93 (4), 307-315 (2013).</li><li>Nudelman, F., Lausch, A. J., Sommerdijk, N. A., Sone, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+collagen+biomineralization.">In vitro models of collagen biomineralization.</a> J. Struct. Biol. 183 (2), 258-269 (2013).</li><li>Alliston, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+regulation+of+bone+quality.">Biological regulation of bone quality.</a> Curr. Osteoporos. Rep. 12 (3), 366-375 (2014).</li><li>Wang, W., Kirsch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+stimulates+annexin-mediated+growth+plate+chondrocyte+mineralization.">Retinoic acid stimulates annexin-mediated growth plate chondrocyte mineralization.</a> J. Cell Biol. 157 (6), 1061-1069 (2002).</li><li>Wang, W., Xu, J., Kirsh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+V+and+terminal+differentiation+of+growth+plate+chondrocytes.">Annexin V and terminal differentiation of growth plate chondrocytes.</a> Exp. Cell Res. 305 (1), 156-165 (2005).</li></ol>

在本文中, 我们描述了 AR 染色的协议, 氟磷灰石的紫外光识别和透射电镜-EDX 的体外成像中的矿化细胞和磁气产生的矿物质.通过遵循一些常见的故障排除步骤, 可以解决上述所有方法。为了获得最佳结果, 应该仔细地执行几个关键步骤。首先, 最好添加 AA (酸性), 其次是β GP (碱性), 以保持培养基的 pH 值7.4。其次, 经过 AR 染色后, 染色的钙沉积物非常脆弱, 细胞应小心清洗, 以防止在添加 ...

骨是由两部分组成的结缔组织: 有机 (细胞和胶原纤维) 和矿物质 (钙和磷酸盐化合物)。骨中主要矿物成分为磷灰石1。不同类型的成矿能力的细胞在骨 (成骨细胞), 在牙齿 (成牙本质细胞) 和软骨 (软骨细胞) 通过产生细胞外基质 (ECM) 和释放基质的蛋白质来调节矿化的初始步骤囊泡 (mv) (图 1)。100-300 毫微米直径囊泡, 积累钙和磷酸促进磷灰石成核, 随后绑定到胶原蛋白2,3。然后, 磷灰石释放到胞外培养基中。磷灰石继续与胶原纤维接触, 形成骨基质。矿化由 Pi和钙2 +在胞外培养基中的恒定供给维持。最近发布的一些数据支持我们的模型4,5。软组织在生理条件下不矿化。然而, 异位钙化可能发生在病理条件下, 如血管钙化3。获得成骨细胞表型的血管细胞可产生诱导磷灰石的核化, 并在血管壁内侧和内膜层启动成矿作用。由于异位钙化类似于正常的软骨矿化3, 了解骨细胞和软骨细胞成矿作用的分子机制, 应为软组织异位钙化提供一些线索。形成。
骨骼组织的发育受各种酶、生长因子、成矿作用的促进剂或抑制剂的调节。组织非特异性碱性磷酸酶 (TNAP) 的拮抗作用 (图 1) 和 ectonucleotide 焦磷酸酶/磷酸二酯化 I (NPP1), 连同锚蛋白 (谢谢), 控制无机焦磷酸盐 (PPI) 浓度6. PPi, 一种有效的 HA 形成抑制剂, 由 TNAP 水解;NPP1 水解核苷酸 triphosphates 形成 ppi , 而谢谢出口 ppi从细胞到 ECM。Pi/PPi 比率可能调控磷灰石形成7,8以可能的病理后果9。
MV 膜富含离子传输蛋白, 在核过程中促进了钙和磷酸盐的初始沉淀 (图 1)。磷酸盐传送器 1 (坑) 帮助合并 Pi生成在 perivesicular 空间入10,11。Annexins 可能涉及 Ca2 +的结合和运输, 以及在 MV 腔12,13中启动矿化的生物物理过程中。我们赞成早些时候提出的假说, 即在其在 ECM14、15中传播之前, 在 MV 内的磷灰石内部成核的胞质囊泡中的矿化。体外建模证实了从 PS 和 AnxA516的 proteoliposomes 中诱导的钙2 +/Pi复合物的形成。这可能表明, 钙2 +, Pi, AnxA5 和 PS 配合物在微绒毛状的 membranesrepresent 中的核核 (NC) 在 M 的 Annexins 和 TNAP也具有胶原结合可能有助于在胶原纤维上放置 mv, 并在促进电解质中的矿化传播方面的能力。牛胎球蛋白 A 和骨桥蛋白 (OPN)17, 被称为磷灰石形成抑制剂, 可能减慢矿化在胶原支架上的传播。成核和传播是不同的事件, 前者前, 两者可能与病理成矿过程有关。
为了发现磷酸钙络合物的化学物质如何改变生理成矿作用和异位钙化, 有必要确定细胞产生的矿物质。磷灰石是一组含有矿物质的钙和磷酸盐, 一般晶体单元细胞配方钙10(PO4)6X2, 其中 X = Cl, F, OH。分类如下18: 氟磷灰石 (FA) 钙10(PO4)6F2, chlorapatite (ca) Ca10(PO4)6Cl2和羟基磷灰石 (HA) 钙10(po4)6(OH)2。
由于每一个细胞系都呈现出明显的矿化特征, 因此选择成骨细胞系诱导矿物形成是至关重要的。在这份报告中, 我们比较了两个选定的人体细胞模型的矿物成核: 成骨 hFOB 1.19 细胞和骨肉瘤 Saos-2 细胞。骨肉瘤衍生细胞通常被用作成骨模型, Saos-2 细胞保存了最成熟的成骨19 , 而未分化的人胎儿 hFOB 细胞作为正常成骨模型被广泛使用。分化20。用不同的方法分析了它们的成矿剖面: 茜素红 s (AR) 染色、紫外 (紫外) 光可视化、透射电镜 (TEM) 成像、能量色散 X 射线微分析 (EDX) 定量和离子映射。EDX 在以往研究中使用的替代技术的优势在于, 它给出了4、5、21磷灰石晶体中离子置换的定量和定性结果。利用 TEM EDX 的总体目标是在成矿过程的不同阶段, 寻找一种简单的方法, 用于成像和定量的各种矿物中的钙、F 和 Cl 离子的分布。该方法成功地用于监测锌纳米粒子与共存化学物质的相互作用及其对水生生物的联合作用22。在另一项研究中, 用电感耦合等离子体发射光谱 (ICP)、N2 储 (注)、XRD、紫外-比 DRS、红外光谱、拉曼光谱等方法, 对钛材料在水溶液中的铜光催化剂进行了广泛的表征。光谱学, 透射电镜 EDX 和光电测量23。我们的目的是比较两个细胞系中的囊泡和矿物质的来源和性质, 以了解在骨分化过程中控制成矿作用的机制。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57423/57423fig1.jpg"> 图 1.从膜中合成细胞外基质 (ECM) 蛋白和释放基质泡 (mv) 的骨细胞中初始化的步骤方案。通过钙结合蛋白、annexins 和磷酸酯的作用, 通过无机磷酸盐转运体 (坑) 的作用, 通过组织非特异碱性磷酸酶 (TNAP) 的活性来积累钙, dephosphorylatesPPi对 Pi, 从而促进磷灰石成核。然后, 磷灰石分解并释放出细胞外培养基。矿化由 Pi和 Ca2 +在胞外介质4,5的恒定供应维持。<a href="https://www.jove.com/files/ftp_upload/57423/57423fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#8217;s DMEM/F12 media mixture</td>
			<td>PAA</td>
			<td>E15-813</td>
			<td>1:1, for human fetus hFOB 1.19 SV40 large T antigen transfected osteoblasts (ATCC CRL-11372)</td>
		</tr>
		<tr>
			<td>McCoy&#8217;s 5A medium</td>
			<td>PAA</td>
			<td>E82312-0025</td>
			<td>for human osteosarcoma Saos-2 cells (ATCC HTB-85)</td>
		</tr>
		<tr>
			<td>Antibiotics mixture (penicillin/streptomycin)</td>
			<td>Sigma</td>
			<td>P0781-100ML</td>
			<td>100 U/mL each</td>
		</tr>
		<tr>
			<td>G-418</td>
			<td>Sigma</td>
			<td>68168</td>
			<td>0.3 mg/mL</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270</td>
			<td>10% for hFOB 1.19 and 15% for Saos-2</td>
		</tr>
		<tr>
			<td>AA</td>
			<td>Sigma</td>
			<td>A-5960</td>
			<td>50 &#181;g/mL</td>
		</tr>
		<tr>
			<td>&#223;-GP</td>
			<td>Sigma</td>
			<td>G9422-100G</td>
			<td>7.5 mM</td>
		</tr>
		<tr>
			<td>Bio-Gel HTP Gel</td>
			<td>Bio-Rad</td>
			<td>130-0420</td>
			<td>for HA</td>
		</tr>
		<tr>
			<td>FA</td>
			<td></td>
			<td></td>
			<td>synthesized by us</td>
		</tr>
		<tr>
			<td>CA</td>
			<td></td>
			<td></td>
			<td>synthesized by us</td>
		</tr>
		<tr>
			<td>Sodium phosphate buffer Na2HPO4/NaH2PO4 mixture</td>
			<td>Sigma</td>
			<td>S7907/S8282</td>
			<td>0.1 M, pH 7.2</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td>pH 7.0, prepared by us</td>
		</tr>
		<tr>
			<td>AR-S in PBS</td>
			<td>Sigma</td>
			<td>A5533-25G</td>
			<td>0.5 g/100 mL, pH 5.0</td>
		</tr>
		<tr>
			<td>Collagenase type IA</td>
			<td>Sigma</td>
			<td>C2674</td>
			<td>500 U/mL</td>
		</tr>
		<tr>
			<td>SCL buffer</td>
			<td></td>
			<td></td>
			<td>prepared by us</td>
		</tr>
		<tr>
			<td>Deionized wather</td>
			<td></td>
			<td></td>
			<td>produced by us</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>POCh</td>
			<td>BA6480111</td>
			<td>absolut 99.8% and solutions 25, 50, 75, 90%</td>
		</tr>
		<tr>
			<td>Uranyl acetate in 50% ethanol</td>
			<td>Polysciences Inc.</td>
			<td>21447-25</td>
			<td>0.25 g/10 mL</td>
		</tr>
		<tr>
			<td>PD medium</td>
			<td></td>
			<td></td>
			<td>pH 7.4, prepared by us</td>
		</tr>
		<tr>
			<td>Fixation mixture (paraformaldehyde/glutaraldehyde)</td>
			<td>Sigma</td>
			<td>158127/G-6257</td>
			<td>3%:1%</td>
		</tr>
		<tr>
			<td>Post-fixation OsO4</td>
			<td>Sigma</td>
			<td>75633</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>LR White resin in ethanol</td>
			<td>Polysciences Inc.</td>
			<td>17411-MUNC 500g</td>
			<td>1:2, 1:1, 100%</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>CHEMPUR</td>
			<td>111024800</td>
			<td>pure</td>
		</tr>
		<tr>
			<td>Tool</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic vials</td>
			<td>Corning Inc.</td>
			<td>430487</td>
			<td>1.2 mL</td>
		</tr>
		<tr>
			<td>Plastic Petri culture dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td>100 mm</td>
		</tr>
		<tr>
			<td>Plastic tubes</td>
			<td>Falcon</td>
			<td>352096 and 352070</td>
			<td>15 and 50 mL</td>
		</tr>
		<tr>
			<td>Serological pipettes</td>
			<td>Falcon and VWR</td>
			<td>357521 and 612-3700</td>
			<td>1 and 10 mL</td>
		</tr>
		<tr>
			<td>Plastic microcentrifuge tubes</td>
			<td>Sigma</td>
			<td>Z688312 and Z628034</td>
			<td>1.5 mL black and 2 mL transparent</td>
		</tr>
		<tr>
			<td>Plastic tips</td>
			<td>VWR</td>
			<td>613-0364, 613-0239 and 613-1050</td>
			<td>0.1-10 &#181;L natural, 1-200 &#181;L yellow and 200-1000 &#181;L blue</td>
		</tr>
		<tr>
			<td>Plastic racks</td>
			<td>Light Labs</td>
			<td>A-7055-Z, A-7053-C</td>
			<td>green for tubes, orange for micro tubes and blue for TEM probes</td>
		</tr>
		<tr>
			<td>Laminar Hera Save</td>
			<td>Thermo Scientific Co.</td>
			<td>KS12</td>
			<td>HEPA filter (H14 according to DIN EN 1822)</td>
		</tr>
		<tr>
			<td>Incubators Hera Cell</td>
			<td>Thermo Scientific Co.</td>
			<td>150</td>
			<td>34&#176;C for hFOB 1.19 and 37&#176;C for Saos-2</td>
		</tr>
		<tr>
			<td>Fume hood</td>
			<td>POLON</td>
			<td>WCS-2</td>
			<td>for TEM stainings</td>
		</tr>
		<tr>
			<td>Glass bottles</td>
			<td>SIMAX</td>
			<td>1632414501050 and 1632414501100</td>
			<td>50 and 100 mL</td>
		</tr>
		<tr>
			<td>Quartz glass tubes</td>
			<td>SIMAX</td>
			<td>638422010100</td>
			<td>&#216; 10 mm, L 100 mm</td>
		</tr>
		<tr>
			<td>Pump</td>
			<td>IBS Integra Biosciences</td>
			<td>VACUSAFE comfort</td>
			<td>for vacuum</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Memmert</td>
			<td>UNE 400</td>
			<td>56&#176;C</td>
		</tr>
		<tr>
			<td>Porcelain multi-well plate</td>
			<td>Rosenthal technik</td>
			<td>229/12</td>
			<td>12 wells</td>
		</tr>
		<tr>
			<td>Glass beakers</td>
			<td>SIMAX</td>
			<td>632417010025</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>Glass bottles</td>
			<td>Pocord</td>
			<td>DIN22</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Plastic box</td>
			<td>Agar Scientific Ltd.</td>
			<td></td>
			<td>for darkness</td>
		</tr>
		<tr>
			<td>Snap Fit Gelatin Capsules</td>
			<td>Agar Scientific Ltd.</td>
			<td>G3741</td>
			<td>size 1</td>
		</tr>
		<tr>
			<td>Formvar/Carbon 300 Mesh Ni grids in box</td>
			<td>Agar Scientific Ltd.</td>
			<td>S162N3</td>
			<td>film on the shiny side</td>
		</tr>
		<tr>
			<td>Silicon cell scraper</td>
			<td>Sigma</td>
			<td>SIAL0010-100EA</td>
			<td>size 1.8/25 cm</td>
		</tr>
		<tr>
			<td>Syringe with needle</td>
			<td>BogMark</td>
			<td>007</td>
			<td>syringe 1 mL 40 U, needle 0.5 x 16</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Chirana</td>
			<td>CH005L</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>MPW Medical Instruments</td>
			<td>MPW-350R</td>
			<td>130 x g and 500 x g</td>
		</tr>
		<tr>
			<td>UV transluminator</td>
			<td>UVP</td>
			<td>M-20</td>
			<td>for visible and UV light</td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>LKB</td>
			<td>NOVA</td>
			<td>700&#197; sections</td>
		</tr>
		<tr>
			<td>Block holder</td>
			<td>LKB</td>
			<td>E6711</td>
			<td>round shape</td>
		</tr>
		<tr>
			<td>Diamond knife</td>
			<td>DiATOME</td>
			<td>Ultra 45&#176;</td>
			<td>size 3</td>
		</tr>
		<tr>
			<td>Eyelash holder</td>
			<td></td>
			<td></td>
			<td>bovine, prepared by us</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>ROTH</td>
			<td>2855.1</td>
			<td>antistatic for grids</td>
		</tr>
		<tr>
			<td>Spatulas set</td>
			<td>ROTH</td>
			<td>E286.1</td>
			<td>antistatic for powders</td>
		</tr>
		<tr>
			<td>Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Light Microscope</td>
			<td>Zeiss with Canon</td>
			<td>AxioObserver Z1 equipped with PowerShot G9</td>
			<td>Phase contrast, Transmitted light, 20 x objective, RGB filters</td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>TEM Jeol Co. with Oxford Instruments and SiS-Olympus</td>
			<td>JEM-1400 TEM equipped with full range INCA Energy Dispersive X-ray microanalysis (EDX) System and 11 Megapixel MORADA G2 camera</td>
			<td>magnification 50,000X for TEM and 15,000X for STEM and EDX</td>
		</tr>
		<tr>
			<td>Camera body and lenses</td>
			<td>Nikon</td>
			<td>Nikon D7100 
			Nikkor AF Micro 105 mm f/2.8D 
			Nikkor AF-S 50 mm f/1.8G 
			Nikkor AF 28 mm f/2.8D
</td>
			<td>for movie recordings</td>
		</tr>
		<tr>
			<td>Microphone</td>
			<td>MXL Mics</td>
			<td>Tempo</td>
			<td>for voice recordings</td>
		</tr>
	</tbody>
</table>

analysis of minerals produced by hfob 1.19 and saos-2 cells using transmission electron microscopy with energy dispersive x-ray microanalysis

本视频介绍了利用透射电镜与能量色散 X 射线微分析 (TEM-EDX) 比较的矿物质在囊泡释放的两个人骨细胞线: hFOB 1.19 和 Saos-2。这些细胞系, 经抗坏血酸 (AA) 和β甘油 (β GP) 治疗后, 经过完全成骨分化, 从增殖到成矿, 产生基质泡 (mv), 触发磷灰石成核在细胞外基质 (ECM)。
基于茜素红 s (AR) 染色, 利用透射电镜成像和 EDX 定量和离子图谱对细胞裂解物中的矿物质组成进行分析, 并对其进行了研究, 得出了 Saos-2 和成骨肉瘤hFOB 1.19 细胞显示出明显的矿化剖面。Saos-2 细胞比 hFOB 1.19 细胞矿化更有效, 并产生较大的矿床, 在紫外光下不可见, 但与羟基磷灰石 (HA) 相似, 因为它们有更多的钙和 F 替代物。
利用这些技术得到的结果使我们得出结论, 矿化过程的不同取决于细胞类型。我们建议, 在细胞层面上, 囊泡的起源和性质预先确定了矿物质的类型。

EDX 允许在体外成像的基质囊泡 (mv) 释放的矿化细胞和矿物生产的 MVs.利用该技术得到的结果表明, 矿化过程可能会有不同的进展。在不同类型的细胞中。两个细胞系接受相同的成骨细胞转分化治疗, 但刺激 Saos-2 单元矿化更有效地比 hFOB 1.19 成骨细胞, 由 AR 染色证明 (图 2)。这可能是由于更成熟的成骨细胞表型的 Saos-2 单元19</...

Watch this Scientific Journal Video about Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis at JoVE.com

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

我们提出一个协议, 以比较的矿物质在囊泡释放的两个人骨细胞系: hFOB 1.19 和 Saos-2。利用茜素红 s (AR) 染色、紫外 (紫外) 光可视化、透射电镜 (TEM) 成像和能量色散 X 射线显微分析 (EDX) 对其成矿剖面进行了研究。

透射电镜 hFOB 1.19 和 Saos-2 细胞产生的能量色散 X 射线显微分析

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

Research

JoVE Journal

Chemistry

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

化学固定准备贴壁细胞的X射线荧光成像

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

科研

化学

Quantifying X-Ray Fluorescence Data Using MAPS

利用地图量化 X 射线荧光数据

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

用于跟踪纳米粒子自组装的液体细胞透射电子显微镜

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

Obtaining 3D Chemical Maps by Energy Filtered Transmission Electron Microscopy Tomography

通过能量过滤透射电镜成像获得3D 化学图

Energy filtered transmission electron microscopy tomography (EFTEM tomography) can provide three-dimensional (3D) chemical maps of materials at a ...

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

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用于原位晶体 x 射线衍射和原位的微流控芯片系列晶体学的动态光散射

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Elemental-sensitive Detection of the Chemistry in Batteries through Soft X-ray Absorption Spectroscopy and Resonant Inelastic X-ray Scattering

软 x 射线吸收光谱和共振非弹性 x 射线散射法检测电池化学成分的元素敏感度

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid ...

Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching

Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching

用石墨烯液细胞透射电镜研究原位纳米晶蚀刻

Graphene liquid cell electron microscopy provides the ability to observe nanoscale chemical transformations and dynamics as the reactions are occurring in ...

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

石墨烯支持的微井液体细胞制备,用于原位透射电子显微镜

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. ...

In situ Grazing Incidence Small Angle X-ray Scattering on Roll-To-Roll Coating of Organic Solar Cells with Laboratory X-ray Instrumentation

通过实验室X射线仪器对有机太阳能电池的滚滚涂层进行现场放牧小角度X射线散射

We present an in-house, in situ Grazing Incidence Small Angle X-ray Scattering (GISAXS) experiment, developed to probe the drying kinetics of roll-to-roll ...

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

通过微透析对芯片上的蛋白质进行结晶，用于 原位 X射线衍射研究

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...