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.
