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 and ASK performed manual operations and LB prepared drawings and made the movie. ASK wrote the manuscript, LB wrote the script and MK prepared the table. SM, RB and SP critically read the table, the script and the manuscript. The authors would like to thank Hanna Chomontowska for her excellent assistance with ultramicrotomy as well as Szymon Suski and Henryk Bilski for their excellent assistance with TEM-EDX analysis. The authors would like to thank dr Patrick Groves for professional English language correction and Barbara Sobiak for recording the instructions.
This work was supported by grant N N401 140639 from the Polish Ministry of Science and Higher Education to ASK, by grants from the National Science Centre, Poland 2016/23/N/NZ4/03313 to LB and 2016/23/N/NZ1/02449 to MK, EU FP7 Project BIOIMAGINE: BIO-IMAGing in research INnovation and Education, GA No. 264173, and by the statutory funds of the Nencki Institute of Experimental Biology, Polish Academy of Sciences.

1. Cell Culture and Treatment
<ol>
	<li>Put all the necessary materials under the laminar flow hood and sterilize them under UV light. The culture media are: a 1:1 mixture of Ham&#39;s F12 and DMEM media with 2.5 mM L-glutamine supplemented with 100 U/mL penicillin, 100 U/mL streptomycin, 0.3 mg/mL G418 and 10% Fetal Bovine Serum (FBS) (v/v) for human fetus hFOB 1.19 SV40 large T antigen transfected osteoblasts, and McCoy&#39;s 5A medium with 1.5 mM L-glutamine supplemented with 100 U/mL penicillin, 100 U/mL streptomyc...

<ol>
	<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>

In the current paper, we described the protocols for AR-S staining, UV light identification of fluorapatite and TEM-EDX in vitro imaging of MVs released by mineralizing cells and of minerals produced by MVs. It is possible to address all methods mentioned above by following some common troubleshooting steps. In order to obtain optimal results, several critical steps should be performed carefully. First, it is better to add AA (which is acidic) followed by &#946;-GP (which is alkaline) to preserve the pH...

Bone is a type of connective tissue composed of two parts: organic (cells and collagen fibers) and mineral (calcium and phosphate compounds). The main mineral components in bones are apatites1. Different types of mineralization-competent cells in bone (osteoblasts), in teeth (odontoblasts) and in cartilage (chondrocytes) regulate the initial steps of mineralization by producing proteins of the extracellular matrix (ECM) and releasing matrix vesicles (MVs) (Figure 1). MVs are 100-300 nm diameter vesicles that accumulate calcium and phosphate facilitating apatite nucleation and subsequently bind to collagen2,3. Then, MVs disintegrate to release apatites to the extracellular medium. The apatites continue to grow in contact with collagen fibers and form the bone matrix. The mineralization is sustained by the constant supply of Pi and Ca2+ in the extracellular medium. Some recently published data support our model4,5. Soft tissues do not mineralize under physiological conditions. However, ectopic calcification may occur under pathological conditions such as vascular calcification3. Vascular cells that acquire the osteoblast phenotype can produce MVs that induce nucleation of apatites and initiate mineralization in the medial and intimal layers of the wall of blood vessels. Since ectopic calcification resemble normal endochondral mineralization3, understanding the molecular mechanisms of mineralization of osseous cells and chondrocytes should provide some clues on ectopic calcification of soft tissues that are formed.
The development of skeletal tissues is regulated by various enzymes, growth factors, and promoters or inhibitors of mineralization. The antagonistic action of tissue-nonspecific alkaline phosphatase (TNAP) (Figure 1) and ectonucleotide pyrophosphatase/phosphodiesterase I (NPP1), together with ankyrin (ANK), controls inorganic pyrophosphate (PPi) concentration6. PPi, a potent inhibitor of HA formation, is hydrolyzed by TNAP; NPP1 hydrolyzes nucleotide triphosphates to form PPi while ANK exports PPi from the cell to the ECM. The Pi/PPi ratio may regulate apatite formation7,8 with possible pathological consequences9.
The MV membrane is enriched in ion transport proteins that facilitate the initial precipitation of calcium and phosphate inside the MVs during the nucleation process (Figure 1). The phosphate transporter 1 (PiT) helps to incorporate Pi generated in the perivesicular space into the MVs10,11. Annexins may be involved in the binding and transport of Ca2+ and in the biophysical process that initiates mineralization in the MV lumen12,13. We favor the hypothesis, suggested earlier, for mineralization within intracytoplasmic vesicles of internal nucleation of apatite inside the MV before its propagation in the ECM14,15. In vitro modeling confirmed the induction of Ca2+/Pi complexes formation in proteoliposomes made from PS and AnxA516. This may indicate that accumulation of Ca2+, Pi, AnxA5 and PS complexes in lipid rafts of microvilli-like membranesrepresent the nucleation core (NC) of apatite within MVs. Annexins and TNAP also possess collagen-binding capacities that may be helpful in placing MVs along collagen fibers and, in stimulating the propagation of mineralization in the ECM. Fetuin A and osteopontin (OPN)17, are known as inhibitors of apatite formation that may slow down the propagation of mineralization on the collagenous scaffold. Nucleation and propagation are distinct events, the former preceding the latter, and both may be relevant for the process of pathological mineralization.
To discover how the chemistry of calcium phosphate complexes may change physiological mineralization and ectopic calcification, it is necessary to identify the minerals produced by cells. Apatites are a group of calcium and phosphate containing minerals with the general crystal unit cell formula Ca10(PO4)6X2, where X = Cl, F, OH. They are classified as follows18: fluorapatite (FA) Ca10(PO4)6F2, chlorapatite (CA) Ca10(PO4)6Cl2 and hydroxyapatite (HA) Ca10(PO4)6(OH)2.
The choice of osteoblast cell lines to induce mineral formation is crucial, since each cell line exhibits a distinct profile of mineralization. In this report, we compared the nucleation of minerals by two selected human cell models of mineralization: osteoblastic hFOB 1.19 cells and osteosarcoma Saos-2 cells. Osteosarcoma-derived cells are commonly used as osteoblastic models and Saos-2 cells have preserved the most mature osteoblastic character19 while undifferentiated human fetal hFOB cells are widely used as a model for normal osteoblastic differentiation20. Their mineralization profiles were analyzed by different methods: Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging, energy dispersive X-ray microanalysis (EDX) quantitation, and ion mapping. The advantage of TEM-EDX over alternative techniques used in previous studies is that it gives quantitative and qualitative results of ion replacement in apatite crystals4,5,21. The overall goal of using TEM-EDX was to find a simple method for imaging and quantification of the distribution of Ca, F and Cl ions in various minerals from different types of cells during distinct stages of the mineralization process. This method has been successfully used, for example, for monitoring the interaction of zinc nanoparticles with coexisting chemicals and their combined effects on aquatic organisms22. In another study, a copper photocatalyst on titanium materials in aqueous solution was extensively characterized by means of inductively coupled plasma optical emission spectrometry (ICP-OES), N2 physisorption (BET), XRD, UV-vis DRS, FT-IR, Raman spectroscopy, TEM-EDX, and photoelectrochemical measurements23. Our aim was to compare the origin and properties of vesicles and minerals in two cell lines to understand the mechanism that controls mineralization during osseous differentiation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57423/57423fig1.jpg" /> 
Figure 1. Scheme of the initial steps of mineralization in osseous cells involving the synthesis of extracellular matrix (ECM) proteins and release of matrix vesicles (MVs) from the membrane. MVs accumulate calcium through the action of calcium binding proteins, annexins and phosphate, through the action of an inorganic phosphate transporter (PiT) followed by the activity of tissue non-specific alkaline phosphatase (TNAP), which dephosphorylates PPi to Pi, thereby facilitating apatite nucleation. Then, MVs disintegrate and release apatites to the extracellular medium. The mineralization is sustained by the constant supply of Pi and Ca2+ in the extracellular medium4,5. <a href="https://www.jove.com/files/ftp_upload/57423/57423fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</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

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and &#946;-glycerophosphate (&#946;-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM).
Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions.
The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

TEM-EDX allows for the in vitro imaging of matrix vesicles (MVs) released by mineralizing cells and of minerals produced by MVs. The results obtained using this technique demonstrate that the process of mineralization may proceed differently in various types of cells. The two cell lines received the same osteoblastic transdifferentiation treatment, yet stimulated Saos-2 cells mineralized more efficiently than hFOB 1.19 osteoblasts, as evidenced by AR-S staining (<strong ...

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

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

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

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

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

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

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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

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