Name: Graphene Coatings for Biomedical Implants (Video) | JoVE Journal
Uploaded: 2013-03-01T13:00:00.000+00:00
Duration: 13 min 21 s
Description: Learn to coat nitinol implants with graphene using chemical vapor deposition for improved hemo compatibility. This method enhances cell viability and morphology in blood-contacting biomedical devices.

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5 minutes

1. Graphene-coating of NiTi
<ol>
 <li>The graphene samples used in this study were grown on copper (Cu) substrates using the chemical vapor deposition technique, and subsequently transferred to 4.5 mm2NiTi substrates.</li>
 <li>Cu foils (1 cm x 1 cm) were placed in a 1 in. quartz tube furnace and heated to 1,000 &deg;C in the presence of 50 sccm of H2 and 450 sccm of Ar.</li>
 <li>Next, methane (1 and 4 sccm) was introduced into the furnace at different flow rates for 20-30 min. The samples were finally cooled to room temperature under flowing H2, Ar and CH4.</li>
 <li>Next, the Cu foils were spin-coated with PMMA (diluted with 4% anisole) at 4,000 rpm followed by a heat treatment for 5 min at 150 &deg;C. Graphene attached to PMMA layer was obtained by etching the Cu foil using Transene Inc., CE-100 etchant, and subsequent rinsing in 10% HCl and de-ionized water for 10 min.</li>
 <li>The samples were transferred to NiTi substrates (4.5 mm2) and annealed at 450 &deg;C in Ar (300 sccm) and H2 (700 sccm) for 2 hr to remove the PMMA. Finally, the substrates were washed with acetone to dissolve the residual PMMA to obtain the Gr-NiTi sample. A Dilor XY triple grating monochromator was used for the micro-Raman characterization (using 100X objective) of all Gr-NiTisamples with the 514.5 nm excitation from an Ar+ ion laser.</li>
</ol>
2. In Vitro Toxicity of Gr-NiTi
Rat aortic endothelial cells (Cell application Inc.,) were cultured on a gelatin coated 8 chambers slide. For testing the cell growth, pristine and Gr-NiTisubstrates were placed in wells without any gelatin coating. Scanning electron microscopy images were obtained using a Hitachi S-4800 SEM. Additionally, rat aortic smooth muscle cells were also grown in CellBind 96-well plates as a control group (Corning) in Dulbecco's Modified Eagle Medium (ATCC). 
<ol>
 <li>For testing cell viability, cells (both endothelial and smooth muscle cells) were seeded at 105 cells/well in wells containing pristine NiTi, 1 sccm or 4 sccm Gr-NiTi substrates, where the stated sccm corresponds to the methane flow used in the CVD growth of graphene. Cells were grown for the desired time period in an incubator at 37 &deg;C and 5% CO2, exchanging media every other day.</li>
 <li>At the end time point, media was removed and fresh media containing 0.5 mg/ml thiazolyl blue tetrazolium bromide (or MTT obtained from Sigma) was added to each well. Cells were then incubated for an additional 3 hr. For the MTS assay, media was removed at the final time point and replaced with 120 &mu;l of MTS working solution (Cell Tier 96 aqueous, Promega) and incubated for 3 hr.</li>
 <li>Next, media was gently removed and 100 ml of dimethylsulfoxide (Sigma) was added to each well. After allowing 10 min for the MTT crystals to dissolve, the solution was transferred to another well plate. For the MTS assay, no dimethylsulfoxide was added to the wells. Well contents were moved to a new plate.</li>
 <li>Absorbance was read at 490 nm and the percent viability was determined by normalizing absorbance to the average absorbance of the pristine NiTi sample. At least five repeats were done for each sample type.</li>
</ol>
3. Confocal Microscopy Studies of Cell Morphology
<ol>
 <li>For confocal imaging of rat aortic smooth muscle cells, substrates were placed in an 8-chamber slide (Thermo Scientific). The cells were seeded at 25,000 cells/chamber and incubated for 3 days at 37 &deg;C and 5% CO2.</li>
 <li>Cells were fixed on the substrate with 4% formaldehyde in phosphate buffered saline for 20 min.</li>
 <li>Permeabilized with 0.1% Triton-X for 1 min.</li>
 <li>Actin was stained with Alexa Fluor 488 phalloidin (Life Technologies). 100 &mu;l of alexafluor 488 phalloidin at 200 units/ml in methanol was added to 1.9 ml of phosphate buffered saline. Cells were stained with 250 &mu;l of alexafluor 488 phalloidin for 45 min and then washed twice with phosphate buffered saline.</li>
 <li>Nuclei were mounted with VectaShield fluorescent mounting medium containing DAPI (Vector Laboratories). Confocal images were collected using a Nikon Confocal TI. A chamber seeded with cells without any substrate was used as a control.</li>
</ol>
4. Protein Adsorption Studies
<ol>
 <li>Substrate dimensions were measured with calipers before starting the protein adsorption experiments. Three measurements were taken for each side of the approximately square samples and averaged to get the length and width.</li>
 <li>Each sample, pristine NiTi, 1sccm and 4sccm Gr-NiTiwere incubated with 1 mg/ml albumin in phosphate buffered saline (PBS) or 1 mg/ml fibrinogen in PBS at room temperature for 3 hr.</li>
 <li>Alike samples were combined in a microcentrifuge tube with 200 &mu;l of reducing sample buffer and boiled for 5 min.</li>
 <li>Samples were then diluted in a Tris/Glycine/SDS buffer (Bio-Rad) and run through a 4-15% Tris polyacrylamide electrophoresis gel (Bio-Rad) at 90 V for 100 min.</li>
 <li>Gels were then stained with SYPRO Red. Dilute SYPRO Red (Life Technologies) stock solution at 1:5,000 in 7.5 v/v% acetic acid. Stain gels for 60 min.</li>
 <li>Image gels using a Flourchem SP (Alpha Innotech).Fluorescent intensity was quantified using ImageJ software. Fluorescent intensity from each sample was normalized by the total area of substrate and fibrinogen adsorption was compared to albumin adsorption.</li>
</ol>
5. Western Blotting for Protein Expression
<ol>
 <li>Western blot was performed to analyze total actin in rat aortic smooth muscle cells. Cells were seeded at 10,000 cells/substrate in a 96-well plate.</li>
 <li>Cells were grown for three days before removing media. Total protein was extracted using RIPA buffer and a standard BCA assay (Lamda) was performed to quantify total protein.</li>
 <li>Samples were diluted to the same concentration in RIPA and then boiled in a reducing sample buffer for 5 min.</li>
 <li>Proteins were separated by a 4-15% Tris polyacrylamide gel via electrophoresis at 90 V for 100 min. A kaleidoscope protein standard (Bio-Rad) was used to assess protein molecular weight.</li>
 <li>Proteins were transferred to a PVDF membrane and blocked with a 1% non-fat dry milk (Bio-Rad) solution.</li>
 <li>Total actin was tagged with a rabbit anti-rat actin antibody (Sigma). A BM chemiluminescent kit (Roche) was used to detect the primary antibody. Membranes were imaged using FlourChem SP imaging equipment and fluorescent intensity was measured using ImageJ software. Fluorescent intensity was normalized by comparing to the pristine NiTisample.</li>
</ol>

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</ol>

No conflicts of interest declared.

Biocompatibility and cytotoxicity: The chemical vapor deposition (CVD) method yielded polycrystalline graphene samples that mimicked Cu crystal grains, as shown in Figure 1a. We employed Raman spectroscopy to confirm the presence of monolayer (few layer) graphene on 1 sccm (4 sccm) samples (see Figure 1b). Clearly, 1 sccm (4 sccm) samples exhibit intense (relatively weaker) G' band indicative of monolayer (few layer) graphene. Figure 1c shows an atomic forc...

The past three decades have witnessed discovery of novel materials-based therapies and devices for disease treatments and diagnostics. Novel alloy materials such as nitinol (NiTi) and stainless steel are often used in biomedical implant manufacturing due to their superior mechanical properties.1-3 However, numerous challenges remain due to exogenous material cytotoxicity, bio- and hemo-compatibility. The metallic nature of these alloys results in poor bio- and hemocompatibility due to metal leaching, lack of cell adhesion, proliferation, and thrombosis when it comes in contact with flowing blood (such as catheters, blood vessel grafts, vascular stents, artificial heart valves etc.). 1, 4, 5 The interaction of proteins or living cells with the implant surface can lead to a strong immunological response and the ensuing cascade of biochemical reactions may adversely affect the device functionality. Therefore, it is pertinent to achieve control over the interactions between biomedical implants and its surrounding biological environment. Surface modification is often employed to reduce or prevent the adverse physiological response originating from the implant material. An ideal surface coating is expected to have high adhesion strength, chemical inertness, high smoothness, and good hemo- and biocompatibility. Previously, numerous materials including diamond-like carbon (DLC), SiC, TiN, TiO2 and many polymer materials have been tested as bio-compatible implant surface coatings. 1, 6-23 However, these materials are still unable to meet all of the functional criteria for a suitable implant surface coating.
The discovery of atom thick layer of sp2 carbon, known as graphene, has opened doors for the development of novel multifunctional materials. Graphene is expected to be an ideal candidate for implant surface coating since it is chemically inert, atomically smooth and highly durable. In this Letter, we investigate the viability of graphene as a surface coating for biomedical implants. Our studies show that the graphene coated nitinol (Gr-NiTi) meets all of the functional criteria, and additionally supports excellent smooth muscle and endothelial cell growth leading to better cell proliferation. We also find that the serum albumin adsorption on Gr-NiTi is higher than fibrinogen. Importantly, (i) our detailed spectroscopic measurements confirmed the lack of charge transfer between graphene and fibrinogen suggesting that graphene coating inhibits platelet activation by implants, (ii) graphene coatings do not exhibit any significant in vitro toxicity for endothelial and smooth muscle cell lines confirming their biocompatibility, and (iii) graphene coatings are chemically inert, durable and impermeable in flowing blood environment. These hemo-and biocompatible properties, along with high strength, chemical inertness and durability, render graphene coatings as an ideal surface coating.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>ATCC</td><td>30-2002</td><td></td></tr><tr><td>Thiazolyl blue tetrazolium bromide</td><td>Sigma-Aldrich</td><td>M2128</td><td></td></tr><tr><td>CellTiter 96 Aqueous One solution cell proliferation assay (MTS)</td><td>Promega</td><td>G3582</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>36.5% formaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>Alexafluor 488 phalloidin</td><td>Life Technologies</td><td>A12379</td><td></td></tr><tr><td>VECTASHIELD mounting medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>Human serum albumin</td><td>Sigma-Aldrich</td><td>A9511</td><td></td></tr><tr><td>Human fibrinogen</td><td></td><td></td><td></td></tr><tr><td>Tris/Glycine/SDS</td><td>Bio-Rad</td><td>161-0732</td><td></td></tr><tr><td>Ready Gel Tris-HCl Gel</td><td>Bio-Rad</td><td>161-1158</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>45726</td><td></td></tr><tr><td>SYPRO Red</td><td>Life Technologies</td><td>S-6653</td><td></td></tr><tr><td>Protein low BCA assay</td><td>Lamda Biotech</td><td>G1003</td><td></td></tr><tr><td>Precision Plus Protein Kaleidoscope Standard</td><td>Bio-Rad</td><td>161-0375</td><td></td></tr><tr><td>Immun-Blot PVDF membrane</td><td>Bio-Rad</td><td>162-0177</td><td></td></tr><tr><td>Blotting grade blocker non-fat dry milk</td><td>Bio-Rad</td><td>170-6404XTU</td><td></td></tr><tr><td>Anti-actin antibody produced in rabbit</td><td>Sigma-Aldrich</td><td>A2066</td><td></td></tr><tr><td>BM Chemiluminescence Western Blotting kit (mouse/rabbit)</td><td>Roche Applied Science</td><td>11520709001</td><td></td></tr><tr><td>RIPA buffer</td><td>Sigma-Aldrich</td><td>R0278</td><td></td></tr><tr><td>NiTi (51% Ni, 49% Ti)</td><td>Alfa-Aesar</td><td>44953</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Horiba JobinYvon</td><td>Raman spectrometer</td><td>Dilor XY 98</td><td></td></tr><tr><td>Nikon</td><td>Confocal microscope</td><td>Eclipse TI microscope</td><td></td></tr><tr><td>Thermoscientific</td><td>Plate reader</td><td></td><td></td></tr><tr><td>Bio-Rad</td><td>Power supply</td><td>164-5050</td><td>PowerPac basic power supply</td></tr><tr><td>Bio-Rad</td><td>Electrophoresis cell</td><td>165-8004</td><td>Mini-PROTEAN tetra cell </td></tr><tr><td>Bio-Rad</td><td>Gel holder cassette</td><td>170-3931</td><td>Mini gel holder cassette</td></tr></tbody></table>

graphene coatings for biomedical implants

Graphene Coatings for Biomedical Implants

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with nanometer thick layers of graphene for applications as a stent material. Graphene was grown on copper substrates via chemical vapor deposition and then transferred onto nitinol substrates. In order to understand how the graphene coating could change biological response, cell viability of rat aortic endothelial cells and rat aortic smooth muscle cells was investigated. Moreover, the effect of graphene-coatings on cell adhesion and morphology was examined with fluorescent confocal microscopy. Cells were stained for actin and nuclei, and there were noticeable differences between pristine nitinol samples compared to graphene-coated samples. Total actin expression from rat aortic smooth muscle cells was found using western blot. Protein adsorption characteristics, an indicator for potential thrombogenicity, were determined for serum albumin and fibrinogen with gel electrophoresis. Moreover, the transfer of charge from fibrinogen to substrate was deduced using Raman spectroscopy. It was found that graphene coating on nitinol substrates met the functional requirements for a stent material and improved the biological response compared to uncoated nitinol. Thus, graphene-coated nitinol is a viable candidate for a stent material.

<img src="/files/ftp_upload/50276/50276fig1.jpg" alt="Graphene synthesis: SEM and Raman spectra, microscopy image shows graphene/NiTi interface." data-altupdatedat="1787630408000" data-alt="Figure 1"> 
 Figure 1. a) CVD grown polycrystalline graphene on Cu foils mimics the metal crystal grains (scale bar: 10 μm). b) Raman spectrum of 1 sccm (4 sccm) graphene shows intense (relatively weaker) G' band indicating monolayer (few layer) nature of a...

Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with ...

The overall goal of this procedure is to demonstrate a method to coat metallic shaped memory alloy nitinol with graphene for improved hemo compatibility in blood contacting implant materials. This is accomplished by first growing graphene on copper foils using chemical vapor deposition. The second step is to etch away the copper foils and transfer the as prepared graphene to nitinol substrates.Next, the graphene coated nitinol substrates are used for culturing endothelial cells and smooth muscle cells. To quantify cyto compatibility, another assay is to check for hemo compatibility by coding both proteins, fibrinogen and albumin onto the graphene coated nitinol substrates. Ultimately, micro ramin spectroscopy, confocal microscopy in vitro cytotoxicity assays and SDS page protein analysis are used to show successful graphene coating, improved cell morphology, improved cell viability, and better hemo compatibility.The main advantage of this technique or existing techniques like polymer coating, is that graphene coatings are extremely durable and corrosion resistant. The implications of this technique extend to origin improvement of stent technologies because graphenes inherent material properties are expected to reduce protein absorption and reduce activation of clotting factors due to protein confirmational change. Though this method can provide insight Into graphene coated nitinol stents can also be used for other systems such as heart valves or catheters.To begin place one centimeter by one centimeter copper foils in a one inch quartz tube furnace and heat to 1000 degrees Celsius in the presence of 50 SCCM of hydrogen and 450 SCC CM of Argonne. Next, introduce one and four SCCM of methane into the 1000 degrees Celsius furnace at different flow rates for 20 to 30 minutes. Cool the samples to room temperature inside the furnace under flowing hydrogen argonne and methane.After removing the samples from the furnace, cut them into 4.5 millimeter squares and use PMMA diluted with 4%Anole to spin coat the copper foils at 4, 000 RPM, followed by a heat treatment for five minutes at 150 degrees Celsius. Etch the copper foil using CE 100 etching until the copper foil is fully etched. Rinse in 10%hydrochloric acid for 10 minutes, then in deionized water for 10 minutes.To obtain the graphene PMMA sample transfer the graphene samples to 4.5 squared millimeter nitinol substrates. Next ail at 450 degrees Celsius in 300 SCCM Argon and 700 SCCM hydrogen for two hours. To remove the PMMA, finally wash the nitinol substrates with acetone to dissolve the residual PMMA to obtain the graphene nickel titanium sample.To begin the toxicity tests, add pristine nitinol one SCCM or four SCCM graphene nitinol substrates to 96 well plates where the stated SCCM corresponds to methane flow used in the chemical vapor deposition growth of graphene. Next seed rat aortic endothelial cells at 10 to the fifth cells per well in some of the wells containing pristine nitinol one SCCM or four SCCM. Graphene nitinol substrates, seed rat aortic smooth muscle cells in a similar manner in a different set of wells.Grow the cells for three days or seven days in an incubator at 37 degrees Celsius and 5%carbon dioxide exchanging media every other day. At the end time point, remove the media from the wells containing smooth muscle cells and add fresh media containing 0.5 milligrams per milliliter MTT to each. Well incubate the cells for an additional three hours.Next, gently remove the media and add 100 microliters of dimethyl sulfoxide to each well. After allowing 10 minutes for the MTT crystals to dissolve, transfer the solution to another well plate. For the MTS assay, remove media from the wells containing endothelial cells at the end time point.Replace with 120 microliters of MTS solution and incubate for three hours. Then transfer the well contents to a new plate. Read the absorbent at 490 nanometers and determine the percent viability by normalizing absorbence to the average absorbance of the pristine nitinol sample.Repeat this step at least five times for each sample. For confocal imaging of rat aortic smooth muscle cells, place the substrates in an eight chamber slide, leaving one chamber empty as a control. Next, seed the cells in all chambers at 25, 000 cells per chamber and incubate for three days at 37 degrees Celsius and 5%carbon dioxide.After removing the media from the slides, adhere the cells on the substrate with 4%formaldehyde in PBS buffer for 20 minutes. Wash with PBS, then permease with 0.1%Triton X for one minute. After washing with PBS, add 100 microliters of the LOR 4 88 PHIN solution to 1.9 milliliters of PBS buffer.To visualize acton stain the cells with 250 microliters of LOR 4 88 Phin for 45 minutes, and then wash twice with PBS buffer. Next, stain the nuclei with a fluorescent mounting medium containing dpi. Collect confocal images using a Nikon confocal ti.Before starting the albumin and fibrinogen protein absorption experiments, measure the substrate dimensions with calipers. Take three measurements for each side of the approximately square samples and average. To get the length and width, place the pristine nitinol sample, the one SCCM Graphene Nitinol sample, and the four SCCM Graphene Nitinol sample into two 12.Well plates add PBS buffer containing one milligram per milliliter albumin. Add room temperature to one of the plates and incubate for three hours separately. Incubate the three sample types with PBS buffer containing one milligram per milliliter.Fibrinogen at room temperature for three hours after washing samples with PBS, combine the like samples in a micro centrifuge tube with 200 microliters of reducing sample buffer and boil for five minutes. Dilute the samples in a one to nine volume to volume ratio of T glycine SDS buffer to deionized water and run through a four to 15%tris poly acrylamide electrophoresis gel at 90 volts for 100 minutes. Dilute spro red stock solution at a one to 5, 000 ratio in 7.5%acetic acid.Next, stain the gels for 60 minutes with the diluted spro red solution. Wrap in aluminum foil to protect the spro red from light image gels using a Fluor chem SP and quantify fluorescent intensity using Image J software. Then normalize the fluorescent intensity from each sample by the total area of substrate and compare fibrinogen absorption to albumin absorption.The chemical vapor deposition method yielded polycrystalline graphene samples that mimic copper crystal grains Ramen spectroscopy was used to confirm the presence of monolayer graphene on one SCCM and four SCCM samples. The one SCCM sample exhibits an intense GPR band indicating the monolayer nature of the as prepared graphene. In contrast, the four SCCM sample shows a relatively weaker GPR band, which confirms the presence of a few layers of graphene pictured here is an atomic force microscopy image of graphene transferred onto nitinol substrates.Detailed measurements of the sample yielded a value of surface roughness of approximately five nanometers.Shown. Here are the confocal optical microscopy images for smooth muscle cells grown on a controlled glass slide on pristine nitinol substrates on one SCCM graphene nitinol substrates, and on four SCCM graphene nitinol substrates. The cells are dense and spherical on both graphene nitinol surfaces, which demonstrates better cell morphology.The MTT and MTS assays show that one and four SCCM graphene nitinol substrates do not exhibit a significant difference in smooth muscle cell viability relative to pristine nitinol. The MTS assays show that the three day cell viability for the endothelial cells was not significantly different than the controls. The scanning electron microscopy images show that the endothelial cells grown on pristine nitinol substrates are sparse and elongated while they're ellipsoidal and dense on the one and four SCCM graphene nitinol substrates.These results demonstrate that graphene coatings lead to better spherical cell morphology of endothelial cells, the one SCCM and four SCCM graphene nitinol samples exhibit low fibrinogen albumin ratio relative to the pristine nitinol sample, suggesting better hemo compatibility arising from graphene. The equilibration of the Fermi level is shown by the energy level diagram for fibrinogen and the density of electronic states For graphene. An electron transfer from fibrinogen to graphene nitinol is only possible from the occupied electronic states of the fibrinogen molecule into empty electronic states of graphene nitinol at the same energy level.Both mono and few layer graphene are semi metals at room temperature with low electron density at the fury level, which results in a weak charge transfer from fibrinogen to graphene. Graphene does not exhibit any changes in the GB band line shape or frequency indicating weak charge transfer from the plasma proteins. The D convoluted peaks obtained from curve fitting are shown in black to demonstrate the usefulness and viability of graphene as an implant coating.A graphene coated copper penny was exposed to 5%hydrogen peroxide. The graphene coated portion of the penny remained unchanged while the uncovered part was discolored by the hydrogen peroxide solution. In addition, graphene nitinol immersed in 70%nitric acid showed no change in the GB band frequency confirming that the graphene coating is extremely durable.Furthermore, the graphene coating in graphene nitinol reduced to the X rate of the underlying copper indicating the impermeability of graphene membranes. After watching this video, you should have a good understanding on how to transfer graphene and biomedical implants to improve their biocompatibility.

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Research

JoVE Journal

Bioengineering

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