After the 3-point bending test, the surfaces of the fractured specimens were evaluated using a stereomicroscope (Stemi SV 11 APO, Carl Zeiss, Oberkochen, Germany). Failure types of specimens were recorded at this stage. The metal-porcelain bonding area of samples randomly selected from 8 groups has been examined with SEM (ZEISS LEO 1430, Carl Zeiss, Oberkochen, Germany) under ×1000 magnifications. Before evaluation of SEM images, specimen surfaces were sputtered with an Au-layer (Polaron SC 7620 Sputter Coater, Quorum Technologies Ltd., Kent, GB).
Polaron sc 7620 sputter coater
Manufactured by Quorum Technologies
Sourced in United Kingdom
The Polaron SC 7620 Sputter Coater is a laboratory equipment used for the deposition of thin films onto samples. The core function of the device is to create a plasma that sputters material from a target onto the surface of the sample, resulting in a thin, uniform coating.
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Lab products found in correlation
Jsm 6400 scanning electron microscope, by JEOL (3 mentions)
Cas 57 50 1, by Merck Group (2 mentions)
Stemi sv 11 apo, by Zeiss (1 mentions)
Leo 1430, by Zeiss (1 mentions)
Fei nova nanosem 430, by Thermo Fisher Scientific (1 mentions)
Cas 6131 99 3, by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
Jsm 6510 scanning electron microscope, by JEOL (1 mentions)
Jsm 5310, by JEOL (1 mentions)
Nova 430, by Thermo Fisher Scientific (1 mentions)
Jsm 6500f, by JEOL (1 mentions)
Scanasyst air probe, by Bruker (1 mentions)
Bioscope resolve afm, by Bruker (1 mentions)
Sodium cacodylate, by Merck Group (1 mentions)
Mono cl4, by Ametek (1 mentions)
Ethanol, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Quanta 450, by Thermo Fisher Scientific (1 mentions)
13 protocols using polaron sc 7620 sputter coater
1
Fracture Analysis of Metal-Porcelain Samples
2
Titanium Surface Evaluation for Cell Attachment
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Titanium sheets were observed under scanning electron microscopy (FEI NOVA NanoSEM 430, FEI Company, Hillsboro, OR, USA) to identify cell attachments on their surface. For SEM analysis, one sample from each group was placed on a sterile 24-well plate and sterilized using ethanol 70% for 30 min. A quantity of 2 × 104 cells/well were cultivated on each titanium sheet at 37 °C in a 5% CO2 for 3 days.
The pre-osteoblasts that adhered to the samples were fixed in a solution of 3% glutaraldehyde (50 wt.% in H2O, CAS#111-30-8, Sigma-Aldrich, San Luis, MO, USA), 0.1 mol/L sodium cacodylate (CAS#6131-99-3, Sigma-Aldrich) and 0.1 mol/L sucrose (CAS#57-50-1, Sigma-Aldrich, San Luis, MO, USA) for 45 min. Samples were immersed for 10 min in a buffer solution of 0.1 mol/L sodium cacodylate (CAS#6131-99-3, Sigma-Aldrich, San Luis, MO, USA) and 0.1 mol/L sucrose (CAS#57-50-1, Sigma-Aldrich, San Luis, MO, USA). Samples were then processed in serial ethanol dehydrations for 10 min each (30, 50, 70 and 100%) and dehydrated in hexamethyldisilazane (HDMS, CAS# 999-97-3, Sigma-Aldrich, San Luis, MO, USA).
Titanium sheets were sputter-coated with a palladium–gold alloy (Polaron SC 7620 Sputter Coater, Quorum Technologies, Laughton, East Sussex, UK) with a thickness of 10 nm. The SEM was operated at 10 kV, spot 3.5 and images were made in 100×, 2000× and 10,000×.
The pre-osteoblasts that adhered to the samples were fixed in a solution of 3% glutaraldehyde (50 wt.% in H2O, CAS#111-30-8, Sigma-Aldrich, San Luis, MO, USA), 0.1 mol/L sodium cacodylate (CAS#6131-99-3, Sigma-Aldrich) and 0.1 mol/L sucrose (CAS#57-50-1, Sigma-Aldrich, San Luis, MO, USA) for 45 min. Samples were immersed for 10 min in a buffer solution of 0.1 mol/L sodium cacodylate (CAS#6131-99-3, Sigma-Aldrich, San Luis, MO, USA) and 0.1 mol/L sucrose (CAS#57-50-1, Sigma-Aldrich, San Luis, MO, USA). Samples were then processed in serial ethanol dehydrations for 10 min each (30, 50, 70 and 100%) and dehydrated in hexamethyldisilazane (HDMS, CAS# 999-97-3, Sigma-Aldrich, San Luis, MO, USA).
Titanium sheets were sputter-coated with a palladium–gold alloy (Polaron SC 7620 Sputter Coater, Quorum Technologies, Laughton, East Sussex, UK) with a thickness of 10 nm. The SEM was operated at 10 kV, spot 3.5 and images were made in 100×, 2000× and 10,000×.
3
Titanium Surface Characterization by SEM
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Titanium sheets were observed under scanning electron microscopy using a MAICE system (JEOL JSM-6400 Scanning Electron Microscope, JEOL LTD, Tokyo, Japan) to identify cell attachments on their surface. The osteoblasts that adhered to the samples were fixed in a solution of 3% glutaraldehyde (50 wt.% in H2O, CAS#111-30-8, Sigma-Aldrich), 0.1 mol/L sodium cacodylate (CAS#6131-99-3, Sigma-Aldrich), and 0.1 mol/L sucrose (CAS#57-50-1, Sigma-Aldrich) for 45 min. Samples were soaked for 10 min in a buffer solution of 0.1 mol/L sodium cacodylate (CAS#6131-99-3, Sigma-Aldrich) and 0.1 mol/L sucrose (CAS#57-50-1, Sigma-Aldrich). Sample surfaces and cells were processed in serial ethanol dehydrations for 10 min each and dehydrated in hexamethyldisilazane (HDMS, CAS# 999-97-3, Sigma-Aldrich) before stored in a desiccator until SEM imaging. Titanium sheets were then sputter-coated with a palladium–gold alloy (Polaron SC 7620 Sputter Coater, Quorum Technologies, Laughton, East Sussex, UK) with a thickness of 10 nm (10–15 mA, under a vacuum of 130 mTorr). After this, the SEM was operated at 5 kV, spot 3 to 6.
The quantitative data were shown as the means ± standard deviations. The statistical differences were calculated using one-way ANOVA and Tukey’s test (GraphPad Prism 9 Software, Inc, San Diego, CA, USA). A p-value of ≤0.05 was considered statistically significant.
The quantitative data were shown as the means ± standard deviations. The statistical differences were calculated using one-way ANOVA and Tukey’s test (GraphPad Prism 9 Software, Inc, San Diego, CA, USA). A p-value of ≤0.05 was considered statistically significant.
4
Native Mass Spectrometry of PI(4,5)P2-Bound Proteins
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Borosilicate nano-electrospray capillaries (Thermo Scientific) were prepared in-house using a P-97 micropipette puller (Sutter Instrument Co.) and coated with palladium/gold in a Polaron SC7620 sputter coater (Quorum Technologies). Native mass spectrometry measurements were done using an Orbitrap Q Exactive Plus UHMR (Thermo Scientific) operated in positive ion mode. Proteins were buffer-exchanged using one or two consecutive Zeba spin desalting columns (Thermo Scientific) into 300 mM ammonium acetate, 1 mM DTT, pH 8. 1 mg di-C8-PI(4,5)P2 (as sodium salt) dissolved in water was added to a mixture of ANTH and ENTH proteins (1:1 molar ratio) to form the complex at a final concentration of 10 µM monomeric proteins and 60 or 200 µM PIP2. Instrument settings were 1.5–1.6 kV capillary voltage, −150 V in source trapping, HCD was off and the AGC target set to 3 × 106 with a maximum inject time of 300 ms. The trapping gas pressure (ratio) was 8, the mass range was 2000–20,000 m/z, and the resolution set to 6250. Raw data were processed and analyzed using UniDec63 (link).
5
Scanning electron microscopy of hAM
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hAM explants were washed in 1x PBS, then fixed in 2.5% glutaraldehyde in PBS (AppliChem, Germany) for 60 min at room temperature and afterwards dehydrated in an ascending ethanol series. After incubating in an ascending hexamethyldisilazane (HMDS, Sigma-Aldrich, USA) series, the membranes were air dried under the fume hood overnight. Then the biopsies were mounted onto double-sided sticky tape on top of an aluminium stub and sputter coated with Pd-Au using a Polaron SC7620 sputter coater (Quorum Technologies Ltd, GB). Afterwards they were analysed using a JEOL JSM-6510 scanning electron microscope (Jeol GmbH, Germany).
6
SEM Membrane Characterization Protocol
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The membranes obtained were dried with air exposure until reaching a constant weight at room temperature (19–22 °C and 65-85% of RH).
The SEM was a Jeol JSM 5310, from Jeol Ltd. (Tokyo, Japan) with Oxford Inca Energy software, version 4.09 (Oxford, UK). Sample preparation was performed by producing a metallic coating with a sputtering Polaron SC 7620 Sputter Coater from Quorum Technologies Ltd, (Laughton UK), with 8-10 mbar of Ar pressure and a target of Au. In these conditions, the coating had a thickness between 150 and 200 Angstroms.
The SEM was a Jeol JSM 5310, from Jeol Ltd. (Tokyo, Japan) with Oxford Inca Energy software, version 4.09 (Oxford, UK). Sample preparation was performed by producing a metallic coating with a sputtering Polaron SC 7620 Sputter Coater from Quorum Technologies Ltd, (Laughton UK), with 8-10 mbar of Ar pressure and a target of Au. In these conditions, the coating had a thickness between 150 and 200 Angstroms.
7
SEM Characterization of Coated Disks
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Non-coated, SiC-coated, and glazed disks were examined under scanning electron microscopy using a MAICE system (JEOL JSM-6400 Scanning Electron Microscope, JEOL LTD, Tokyo, Japan) to characterize surface roughness. After the coated and non-coated disks were incubated for 24 h, the culture medium was removed, and the polymicrobial biofilm adhered to the samples was fixed in a solution of 3% glutaraldehyde, 0.1 mol/L sodium cacodylate, and 0.1 mol/L sucrose for 45 min. Samples were soaked for 10 min in a buffer solution of 0.1 mol/L sodium cacodylate and 0.1 mol/L sucrose. Sample surfaces and cells were processed in serial ethanol dehydrations for 10 min each and dehydrated in hexamethyldisilazane (HDMS) before being stored in a desiccator until SEM imaging. The samples were then sputter-coated with a palladium–gold alloy (Polaron SC 7620 Sputter Coater, Quorum Technologies, Laughton, East Sussex, United Kingdom) with a thickness of 10 nm to reduce charging effects during SEM analysis (10–15 mA, under a vacuum of 130 mTorr). After this, the SEM was operated at 5 kV, spot 3 to 6 (FEI NOVA 430, Hillsboro, OR, USA).
8
Surface Topography and Roughness Analysis
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AFM was performed to evaluate surface topography and roughness of the PEMs, on a Bioscope Resolve AFM (Bruker) with ScanAsyst Air probes. The measurements were performed under ambient conditions, room temperature in air at 1 Hz line rate. AFM images were obtained using tapping mode from a scanning image probe processor version 4.2.2.0 software. For SEM, the samples were coated with palladium–gold alloy (Polaron SC 7620 Sputter Coater, Quorum Technologies, Newhaven, UK) at a thickness of 10 nm (10–15 mA, under a vacuum of 130 mTorr). The SEM (JSM-6500F, field emission scanning electron microscope, JEOL, Japan) was operated at an accelerating voltage of 5 kV.
9
Quantifying Microbial Biofilm Adhesion on Titanium
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After 30 days, the biofilm adhesion on the Ti sheets surface were identified under scanning electron microscopy using a MAICE system (JEOL JSM-6400 Scanning Electron Microscope, JEOL LTD, Tokyo, Japan). The monomicrobial biofilm adhering to the samples was fixed in a solution of 3% glutaraldehyde (50 wt.% in H2O, CAS#111-30-8, Sigma-Aldrich), 0.1 mol/L sodium cacodylate (CAS#6131-99-3, Sigma-Aldrich), and 0.1 mol/L sucrose (CAS#57-50-1, Sigma-Aldrich) for 45 min. Samples were placed in a buffer solution of 0.1 mol/L sucrose (CAS#57-50-1, Sigma-Aldrich) and 0.1 mol/L sodium cacodylate (CAS#6131-99-3, Sigma-Aldrich) for 10 min. Ti sheets surface and biofilm were treated in serial ethanol dehydrations for 10 min each and dehydrated in hexamethyldisilazane (HDMS, CAS# 999-97-3, Sigma-Aldrich) until SEM imaging. Ti sheets were then sputter-coated with a palladium–gold alloy (Polaron SC 7620 Sputter Coater, Quorum Technologies, Laughton, East Sussex, UK) with a thickness of 10 nm to reduce charging effects during SEM analysis (10–15 mA, under a vacuum of 130 mTorr). After this, the SEM was operated at 5 kV, spot 3 to 6.
In addition, SEM images of the biofilm on the surface of the Ti sheets were analyzed by ImageJ software. Bacteria coverage percentages were averaged over five random areas.
In addition, SEM images of the biofilm on the surface of the Ti sheets were analyzed by ImageJ software. Bacteria coverage percentages were averaged over five random areas.
10
Bacterial Adhesion on Titanium Surfaces
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The coated and uncoated titanium groups were examined using an SEM microscope (FEI Nova 430, JEOL, Hillsboro, OR, USA) to determine the bacterial adhesion. After the coated and uncoated disks were incubated, the culture medium was removed, and the monomicrobial biofilm adhering to the samples was placed in a fixation solution of 3% glutaraldehyde, 0.1 mol/L sodium cacodylate, and 0.1 mol/L sucrose for 45 min. The samples were soaked for 10 min in a buffer solution (0.1 mol/L sodium cacodylate and 0.1 mol/L sucrose). The sample surfaces and biofilm were treated in serial ethanol dehydrations for 10 min each, dehydrated in hexamethyldisilazane (HDMS), and stored in a desiccator until SEM imaging. Then, the samples were sputter-coated with a palladium-gold alloy (Polaron SC 7620 Sputter Coater, Quorum Technologies, Lewes, UK) to reduce charging effects during the analysis.
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