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>
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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 ...

During the initial steps of mineralization, cells produce extracellular matrix proteins and release matrix vesicles that accumulate calcium and phosphate to facilitate apatite nucleation. In this report, we compare the nucleation of minerals by two selected human cell models, osteoblastic hFOB 1.19 and osteosarcoma Saos-2 cells. Their mineralization profiles were analyzed by different methods.Put all materials under the laminar, and sterilize them under UV light. Change the medium to 10 milliliters of fresh culture medium with fetal bovine serum. Resting cells are incubated without stimulators, whereas stimulated cells are treated with both ascorbic acid and beta-glycerophosphate.First, gently drop ascorbic acid to final concentration of 50 micrograms per milliliter onto the surface of the culture medium to stimulate the cells. Next, add beta-glycerophosphate to final concentration of 7.5 millimolar in culture medium to stimulate the cells. Gently stir the culture dish, and put it into the incubator for seven days.After seven days of incubation, wash the cell cultures with phosphate buffered saline, PBS. Add five milliliters of 2%alizarin red, and incubate the plate for 30 minutes to stain the minerals. Wash three times with PBS.Carefully add PBS to the dish wall. Try not to destroy the minerals. Observe the minerals under an inverted light microscope.Collect the medium from cell cultures, and wash the cells with PBS. Digest either resting or stimulated-for-seven-days cells with 500 units per milliliter collagenase solution at 37 degrees for three hours. Then, mechanically scrape the cells.Transfer them to plastic microcentrifuge tubes. Pass them through a syringe 10 times. Centrifuge the samples at 500 g for five minutes.Discard the supernatant, and suspend the cell pellet in 500 microliters of synthetic cartilage lymph. Transfer the hydroxyapatite, fluorapatite, and chlorapatite powders from the bottles on the UV transilluminator with spatula, and use as controls. Transfer the cell lysates from the plastic tubes with plastic tips, and place carefully on the UV transilluminator.Suspend 2.5 milligrams of hydroxyapatite, fluorapatite, and chlorapatite powders in 500 microliters of deionized water. Incubate at 37 degrees for an hour. Take Formvar/carbon nickel grids from the box with antistatic forceps, and place it on a porcelain multi-well plate.Drop 10 microliters of the hydroxyapatite suspension on the grids. Do the same for chlorapatite and fluorapatite. Dry the samples for 30 minutes at room temperature.Prepare resting and stimulated cells for embedding. Collect medium from the cell cultures. Wash the cells in physiological desensitization medium.Fix the cells with five milliliters of a mixture of 3%paraformaldehyde and 1%glutaraldehyde in 100 millimolar sodium phosphate buffer for an hour at room temperature under the fume hood. Wash the cells with five milliliters of 100 millimolar sodium phosphate buffer. Gently remove the buffer after washing.In the dark room, postfix the samples with two milliliters of 1%osmium tetroxide in 100 millimolar sodium phosphate buffer. Incubate the samples for 20 minutes at room temperature under the fume hood. Remove osmium tetroxide, and utilize it.Wash the cells with five milliliters of 100 millimolar sodium phosphate buffer. Then, dehydrate the samples in five milliliter aliquots of a graded ethanol solution series at room temperature, 25, 50, 75, and 90%ethanol. Finally, use absolute ethanol twice, and incubate for 30 minutes and 12 hours.Mechanically scrape the cells from the culture dishes. Collect the cells into plastic microcentrifuge tubes. Centrifuge the samples at 130 g for one minute.Remove the supernatants. Suspend the cells in a mixture of LR White resin and absolute ethanol at a volume ratio of one to two. Mix well, and incubate for 30 minutes at room temperature.Centrifuge the samples at 130 g for one minute. Remove the supernatants. Repeat the previous step using a one-to-one mixture of LR White resin and absolute ethanol.Mix well, and incubate for 30 minutes at room temperature. Centrifuge the samples at 130 g for one minute. Remove the supernatants.Add pure LR White resin to the samples, and incubate for one hour at room temperature in plastic tubes. Centrifuge the samples at 130 g for one minute. Remove the supernatants.Add again pure LR White resin. Place 500 microliters of each sample into gelatin capsules. Note, the samples are labeled using a small sheet of paper and a pencil so that the resin does not destroy the labels.Close the gelatin capsules. Put the capsules into plastic microcentrifuge tubes. Centrifuge at 130 g for one minute in a swing-out rotor.Remove the capsules from the plastic tubes using a vacuum pump. Move the samples to the oven, and polymerize at 56 degrees for 48 hours. Prepare the blocks by mounting them into the holder.Put the holder into the ultramicrotome. Attach the diamond knife. Fill it with deionized water.Remember, clean the blade from the incidental air bubbles. Then, cut sections using a diamond knife on the deionized water bath. Set the scraps using bovine eyelash.Then, place them on the shiny side of the Formvar/carbon nickel grid, and dry them. In the dark room, counterstain the grids of synthetic apatites and cell samples with 2.5%uranyl acetate in ethanol for 20 minutes at room temperature under fume hood. Wash the grids in 50%ethanol, then in deionized water, and dry it at room temperature for 24 hours.Finally, put the grids into the box. Prepare the beryllium holder for the observation of minerals and cells. Use antistatic tools.Remove the pair of screws. Lift the beryllium plate and beryllium washer away from the rest of the retainer. Mount the grid, shiny side up, on the holder.Carefully place the beryllium washer and beryllium plate. Screw tightly the fastening screws. Put the holder into the vacuum chamber, and turn on the vacuum pump.Once a vacuum is achieved, gently insert the holder into the imaging chamber and turn on the beam. On the fluorescent monitor, set the aperture parameters of the microscope. Perform image astigmatism correction.Set the zoom, focus, frame, and take a transmission electron microscopy image. Move the detector into the imaging chamber. Adjust the sharpness of the image in the focus mode.Select a point in the sample for X-ray microanalysis, and collect spectra. Select elements such as calcium, fluorine, chlorine, and phosphorus to make ion mapping and perform perform maps of the selected elements. Stimulated Saos cells mineralized more efficiently than hFOB osteoblasts.Only fluorapatites can be observed under UV light excitation. HFOB cell lysates were more fluorescent than Saos, suggesting that fluorapatites are more abundant in these cells. Stimulated Saos cells produce more vesicles containing minerals compared to hFOB cells and resting Saos cells.Synthetic apatites had different mineral forms. The procedure applied above showed the presence of vesicles in hFOB and Saos cells under resting and stimulated conditions. The red points indicate the calcium, green phosphorus, and blue fluorine distributions.Under stimulated conditions, there was a strong overlap between calcium and phosphorus distributions in vesicles produced by Saos cells and between fluorine and phosphorus in vesicles produced by hFOB cells. In conclusion, our findings indicate that the vesicles are key determinants of mineral nucleation, especially at the cellular level. The comparison of vesicles released from Saos-2 cells, which mineralize more efficiently, and vesicles released from hFOB 1.19 cells support the hypothesis that matrix vesicles are mineral-filled vesicles.This leaves open the question whether the presence of matrix vesicles is required to induce apatite nucleation and how this may alter physiological to pathological mineralization.

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