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>

<ol>
	<li>Roy, R. K., Lee, K. -. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+applications+of+diamond-like+carbon+coatings:+A+review.">Biomedical applications of diamond-like carbon coatings: A review.</a> Journal of Biomedical Materials Research Part B-Applied Biomaterials. 83 B (1), 72-84 (2007).</li><li>Shah, A. K., Sinha, R. K., Hickok, N. J., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+morphometric+analysis+of+human+osteoblastic+cell+adhesion+on+clinically+relevant+orthopedic+alloys.">High-resolution morphometric analysis of human osteoblastic cell adhesion on clinically relevant orthopedic alloys.</a> Bone. 24 (5), 499-506 (1999).</li><li>Huang, N., Yang, P., Leng, Y. X., Chen, J. Y., Sun, H., Wang, J., Wang, G. J., Ding, P. D., Xi, T. F., Leng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemocompatibility+of+titanium+oxide+films.">Hemocompatibility of titanium oxide films.</a> Biomaterials. 24 (13), 2177-2187 (2003).</li><li>Gutensohn, K., Beythien, C., Bau, J., Fenner, T., Grewe, P., Koester, R., Padmanaban, K., Kuehnl, P. <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+analyses+of+diamond-like+carbon+coated+stents:+Reduction+of+metal+ion+release,+platelet+activation,+and+thrombogenicity.">In vitro analyses of diamond-like carbon coated stents: Reduction of metal ion release, platelet activation, and thrombogenicity.</a> Thrombosis Research. 99 (6), 577-585 (2000).</li><li>Gillespie, W. J., Frampton, C. M. A., Henderson, R. J., Ryan, P. 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+Incidence+of+Cancer+Following+Total+Hip-Replacement.">The Incidence of Cancer Following Total Hip-Replacement.</a> Journal of Bone and Joint Surgery-British Volume. 70 (4), 539-542 (1988).</li><li>Sperling, C., Schweiss, R. B., Streller, U., Werner, C. <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+hemocompatibility+of+self-assembled+monolayers+displaying+various+functional+groups.">In vitro hemocompatibility of self-assembled monolayers displaying various functional groups.</a> Biomaterials. 26 (33), 6547-6557 (2005).</li><li>Mikhalovska, L. I., Santin, M., Denyer, S. P., Lloyd, A. W., Teer, D. G., Field, S., Mikhalovsky, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinogen+adsorption+and+platelet+adhesion+to+metal+and+carbon+coatings.">Fibrinogen adsorption and platelet adhesion to metal and carbon coatings.</a> Thrombosis and Haemostasis. 92 (5), 1032-1039 (2004).</li><li>Airoldi, F., Colombo, A., Tavano, D., Stankovic, G., Klugmann, S., Paolillo, V., Bonizzoni, E., Briguori, C., Carlino, M., Montorfano, M., Liistro, F., Castelli, A., Ferrari, A., Sgura, F., Mario, C. D. i. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+diamond-like+carbon-coated+stents+versus+uncoated+stainless+steel+stents+in+coronary+artery+disease.">Comparison of diamond-like carbon-coated stents versus uncoated stainless steel stents in coronary artery disease.</a> American Journal of Cardiology. 93 (4), 474-477 (2004).</li><li>Allen, M., Myer, B., Rushton, N. <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+and+in+vivo+investigations+into+the+biocompatibility+of+diamond-like+carbon+(DLC)+coatings+for+orthopedic+applications.">In vitro and in vivo investigations into the biocompatibility of diamond-like carbon (DLC) coatings for orthopedic applications.</a> Journal of Biomedical Materials Research. 58 (3), 319-328 (2001).</li><li>Butter, R., Allen, M., Chandra, L., Lettington, A. H., Rushton, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In-vitro Studies of DLC Coatings with Silicon Intermediate Layer. Diamond and Related Materials. 4 (5-6), 857-861 (1995).</li><li>Dearnaley, G., Arps, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+applications+of+diamond-like+carbon+(DLC)+coatings:+A+review.">Biomedical applications of diamond-like carbon (DLC) coatings: A review.</a> Surface &amp; Coatings Technology. 200 (7), 2518-2524 (2005).</li><li>Dorner-Reisel, A., Schurer, C., Nischan, C., Seidel, O., Muller, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diamond-like+carbon:+alteration+of+the+biological+acceptance+due+to+Ca-O+incorporation.">Diamond-like carbon: alteration of the biological acceptance due to Ca-O incorporation.</a> Thin Solid Films. 420, 263-268 (2002).</li><li>Dowling, D. P., Kola, P. V., Donnelly, K., Kelly, T. C., Brumitt, K., Lloyd, L., Eloy, R., Therin, M., Weill, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+diamond-like+carbon-coated+orthopaedic+implants.">Evaluation of diamond-like carbon-coated orthopaedic implants.</a> Diamond and Related Materials. 6 (2-4), 390-393 (1997).</li><li>Grill, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diamond-like+carbon+coatings+as+biocompatible+materials+-+an+overview.">Diamond-like carbon coatings as biocompatible materials - an overview.</a> Diamond and Related Materials. 12 (2), 166-170 (2003).</li><li>Hauert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+modified+DLC+coatings+for+biological+applications.">A review of modified DLC coatings for biological applications.</a> Diamond and Related Materials. 12 (3-7), 583-589 (2003).</li><li>Windecker, S., Mayer, I., De Pasquale, G., Maier, W., Dirsch, O., De Groot, P., Wu, Y. P., Noll, G., Leskosek, B., Meier, B., Hess, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+Grp+Novel+Surface,+C.,+Stent+coating+with+titanium-nitride-oxide+for+reduction+of+neointimal+hyperplasia.">Working Grp Novel Surface, C., Stent coating with titanium-nitride-oxide for reduction of neointimal hyperplasia.</a> Circulation. 104 (8), 928-933 (2001).</li><li>Zhang, F., Zheng, Z. H., Chen, Y., Liu, X. G., Chen, A. Q., Jiang, Z. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+investigation+of+blood+compatibility+of+titanium+oxide+films.">In vivo investigation of blood compatibility of titanium oxide films.</a> Journal of Biomedical Materials Research. 42 (1), 128-133 (1998).</li><li>Bolz, A., Schaldach, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial-Heart+Valves+-+Improved+Blood+Compatibility+By+PECVD+a-SiC-H+COATING.">Artificial-Heart Valves - Improved Blood Compatibility By PECVD a-SiC-H COATING.</a> Artificial Organs. 14 (4), 260-269 (1990).</li><li>Haude, M., Konorza, T. F. M., Kalnins, U., Erglis, A., Saunamaki, K., Glogar, H. D., Grube, E., Gil, R., Serra, A., Richardt, H. G., Sick, P., Erbel, R., Invest, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin-coated+stent+placement+for+the+treatment+of+stenoses+in+small+coronary+arteries+of+symptomatic+patients.">Heparin-coated stent placement for the treatment of stenoses in small coronary arteries of symptomatic patients.</a> Circulation. 107 (9), 1265-1270 (2003).</li><li>Suggs, L. J., Shive, M. S., Garcia, C. A., Anderson, J. M., Mikos, A. G. <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+cytotoxicity+and+in+vivo+biocompatibility+of+poly(propylene+fumarate-co-ethylene+glycol)+hydrogels.">In vitro cytotoxicity and in vivo biocompatibility of poly(propylene fumarate-co-ethylene glycol) hydrogels.</a> Journal of Biomedical Materials Research. 46 (1), 22-32 (1999).</li><li>Clarotti, G., Schue, F., Sledz, J., Benaoumar, A. A., Geckeler, K. E., Orsetti, A., Paleirac, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+the+biocompatible+and+haemocompatible+properties+of+polymer+substrates+by+plasma-deposited+fluorocarbon+coatings.">Modification of the biocompatible and haemocompatible properties of polymer substrates by plasma-deposited fluorocarbon coatings.</a> Biomaterials. 13 (12), 832-840 (1992).</li><li>Gombotz, W. R., Guanghui, W., Horbett, T. A., Hoffman, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+adsorption+to+poly(ethylene+oxide)+surfaces.">Protein adsorption to poly(ethylene oxide) surfaces.</a> Journal of Biomedical Materials Research. 25 (12), 1547-1562 (1991).</li><li>Ishihara, K., Fukumoto, K., Iwasaki, Y., Nakabayashi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+polysulfone+with+phospholipid+polymer+for+improvement+of+the+blood+compatibility.+Part+2.+Protein+adsorption+and+platelet+adhesion.">Modification of polysulfone with phospholipid polymer for improvement of the blood compatibility. Part 2. Protein adsorption and platelet adhesion.</a> Biomaterials. 20 (17), 1553-1559 (1999).</li><li>Jung, N., Kim, B., Crowther, A. C., Kim, N., Nuckolls, C., Brus, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Reflectivity+and+Raman+Scattering+in+Few-Layer-Thick+Graphene+Highly+Doped+by+K+and+Rb.">Optical Reflectivity and Raman Scattering in Few-Layer-Thick Graphene Highly Doped by K and Rb.</a> ACS Nano. 5 (7), 5708-5716 (2011).</li><li>Rao, A. M., Eklund, P. C., Bandow, S., Thess, A., Smalley, 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=Evidence+for+charge+transfer+in+doped+carbon+nanotube+bundles+from+Raman+scattering.">Evidence for charge transfer in doped carbon nanotube bundles from Raman scattering.</a> Nature. 388 (6639), 257-259 (1997).</li><li>Bunch, J. S., Verbridge, S. S., Alden, J. S., vander Zande, A. M., Parpia, J. M., Craighead, H. G., McEuen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impermeable+atomic+membranes+from+graphene+sheets.">Impermeable atomic membranes from graphene sheets.</a> Nano Letters. 8 (8), 2458-2462 (2008).</li><li>Chen, S., Brown, L., Levendorf, M., Cai, W., Ju, S. -. Y., Edgeworth, J., Li, X., Magnuson, C. W., Velamakanni, A., Piner, R. D., Kang, J., Park, J., Ruoff, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+Resistance+of+Graphene-Coated+Cu+and+Cu/Ni+Alloy.">Oxidation Resistance of Graphene-Coated Cu and Cu/Ni Alloy.</a> Acs Nano. 5 (2), 1321-1327 (2011).</li></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 border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Dulbecco'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></td>
 <td></td>
 <td></td>
 <td>Equipment</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

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="Figure 1"/> 
 Figure 1. a) CVD grown polycrystalline graphene on Cu foils mimics the metal crystal grains (scale bar: 10 &mu;m). b) Raman spectrum of 1 sccm (4 sccm) graphene shows intense (relatively weaker) G' band indicating monolayer (few layer) nature of as-prepared graphene. c) AFM image of graphene transferred on to NiTi shows a roughness of ~5 nm. Scale bar = 500 nm.
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Watch this Scientific Journal Video about Graphene Coatings for Biomedical Implants at JoVE.com

Graphene Coatings for Biomedical Implants

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 ...

graphene-coatings-for-biomedical-implants

Research

JoVE Journal

Bioengineering

21.2K Views.  Clemson University. Графен предлагает потенциал в качестве материала покрытия для биомедицинских имплантатов. В этом исследовании мы демонстрируем метод для покрытия сплавов нитинола с нанометровой толщины слоев графена и определить, как графен может повлиять на имплантат от?...

Video: Graphene Coatings for Biomedical Implants

Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very promising application for molecular sensors, field effect transistor and super thin and flexible electronic devices1-4. Anodic arc discharge supported by the erosion of the anode material is one of the most practical and efficient methods, which can provide specific non-equilibrium processes and a high influx of carbon material to the developing structures at relatively higher temperature, and consequently the as-synthesized products have few structural defects and better crystallinity.
 To further improve the controllability and flexibility of the synthesis of carbon nanostructures in arc discharge, magnetic fields can be applied during the synthesis process according to the strong magnetic responses of arc plasmas. It was demonstrated that the magnetically-enhanced arc discharge can increase the average length of SWCNT 5, narrow the diameter distribution of metallic catalyst particles and carbon nanotubes 6, and change the ratio of metallic and semiconducting carbon nanotubes 7, as well as lead to graphene synthesis 8. 
 Furthermore, it is worthwhile to remark that when we introduce a non-uniform magnetic field with the component normal to the current in arc, the Lorentz force along the J&times;B direction can generate the plasmas jet and make effective delivery of carbon ion particles and heat flux to samples. As a result, large-scale graphene flakes and high-purity single-walled carbon nanotubes were simultaneously generated by such new magnetically-enhanced anodic arc method. Arc imaging, scanning electron microscope (SEM), transmission electron microscope (TEM) and Raman spectroscopy were employed to analyze the characterization of carbon nanostructures. These findings indicate a wide spectrum of opportunities to manipulate with the properties of nanostructures produced in plasmas by means of controlling the arc conditions.

Anodic arc discharge is one of the most practical and efficient methods to synthesize various carbon nanostructures. To increase the arc controllability and flexibility, a non-uniform magnetic field was introduced to process the one-step synthesis of large-scale graphene flakes and high-purity single-walled carbon nanotubes.

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb monoliths (MHMs) with micrometer-scale channels are expected as efficient platforms for reactions and separations because of their large surface areas. Up to now, MHMs have been prepared by a unidirectional freeze-drying (UDF) method only from very limited precursors. Herein, we report a protocol from which a series of MHMs consisting of different components can be obtained. Recently, we found that cellulose nanofibers function as a distinct structure-directing agent towards the formation of MHMs through the UDF process. By mixing the cellulose nanofibers with water soluble substances which do not yield MHMs, a variety of composite MHMs can be prepared. This significantly enriches the chemical constitution of MHMs towards versatile applications.

Here, we present a general protocol to prepare a variety of microhoneycomb monoliths (MHMs) in which fluid can pass through with an extremely low pressure drop. MHMs obtained are expected to be used as filters, catalyst supports, flow-type electrodes, sensors and scaffolds for biomaterials.

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior of these materials. In the past, it has not been possible to satisfactorily measure the softening of thin films under conditions mimicking body environment without substantial effort. This publication presents a new and simple method that allows dynamic mechanical analysis (DMA) of polymers in solutions, such as phosphate buffered saline (PBS), at relevant temperatures. The use of environmental DMA allows measurement of the softening effects of polymers due to plasticization in various media and temperatures, which therefore allows a prediction of the materials behavior under in vivo conditions.

To allow reliable predictions of the softening of polymeric substrates for neural implants in an in vivo environment, it is important to have a reliable in vitro method. Here, the use of dynamic mechanical analysis in phosphate buffered saline at body temperature is presented.

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different species. The octopus is one of these exceptional animals that has given scientists new insights into the fields of neuroscience, robotics, regenerative medicine, and prosthetics. Research with this species of cephalopods requires the set-up of complex facilities and intensive care for both the octopus and its ecosystem that is critical for the project&#39;s success. This system requires multiple mechanical and biological filtering systems to provide a safe and clean environment for the animal. Along with the control system, specialized routine maintenance and cleaning are required to effectively keep the facility operating long term. It is advised to provide an enriched environment to these intelligent animals by changing the tank&#39;s landscape, incorporating a variety of prey, and introducing challenging tasks for them to work through. Our results include MRI and a whole-body autofluorescence imaging as well as behavioral studies to better understand their nervous system. Octopuses possess unique physiology that can impact many areas of biomedical research. Providing them with a sustainable ecosystem is the first crucial step in uncovering their distinct capabilities.

Understanding the unique physiological and anatomical structures of octopuses can greatly impact biomedical research. This guide demonstrates how to set-up and maintain a marine environment to accommodate this species and includes state-of-the-art imaging and analytical approaches to visualize octopus' nervous system anatomy and function.

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different ...