The overall goal of this procedure is to synthesize bacterial nano-cellulose, or BNC, and render it magnetic by impregnating in situ with iron oxide nano-particles. Magnetic BNC membranes are designed to locally attact and retain magnetized cells against hemodynamic flow. The main advantage of the magnetic BNC synthesized in this procedure is that it provides a minimal invasive treatment near brain aneurysms.
Convention treatments of brain aneurysms such as clipping, and neural and vascular core emulisation are invasive and traumatic and not suitable in most patients with increased risks. The implications of this technique extend towards therapy of vascular diseases, because vascular grafts, made from magnetic scaffolds can speed healing by improving cell retention at the site of injury. Begin this procedure with preparation of the liquid culture medium as described in the text protocol.
Transfer two milliliters of the liquid culture medium into each well of a 24 well tissue culture plate. Take two colonies with an inoculating needle from the inoculated petri dishes in step, and place them into the first well of the tissue culture plate. Repeat the same procedure for the remaining 23 wells.
Incubate the tissue culture plate at 30 degrees Celsius for seven days. This will yield a total of 24 BNC pellicles with a diameter of 16 mm and a thickness of approximately 2 to 3 mm. Collect the BNC pellicles from the growth media and sterilize them in 200 milliliters of one percent sodium hydroxide solution for one hour at 50 degrees Celsius to remove all traces of G.Xylinus.
Discard the solution, and add 200 milliliters of freshly prepared one percent sodium hydroxide solution. Repeat the same process once more or until the BNC pellicles in solution acquire a translucent appearance. Rinse the BNC pellicles with water three times, and store them in high purity water at room temperature.
Make sure the BNC pellicles are completely submerged in the water and are not allowed to dry at any time. Then, autoclave the BNC pellicles at 121 degrees Celsius for 20 minutes. To begin synthesis, bubble 1000 milliliters of high purity water with nitrogen gas to remove any dissolved oxygen in the water and replace it with nitrogen.
Use a three neck round-bottom flask to prepare a solution in a 2 to 1 molar ratio of iron 3 chloride hexahydrate and iron 2 chloride tetra-hydrate, diluted with the de-oxygenated high purity water. Use 2 necks of the vessel to provide a constant entrance and output of nitrogen gas by connecting the nitrogen gas supply to a needle punched in a septum stopper and fixed to the vessel's neck. Place one BNC pellicle in the vessel with the reactants.
Make sure the sample is completely submerged in the liquid. Then, fill all of the glass joints with vacuum grease. Connect the remaining neck of the vessel to a condenser tube topped with a drying tube filled with anhydrous calcium sulphate.
Run water through the condenser tube. Next, heat the solution in a silicone oil bath to 80 degrees Celsius, using a stirring hot plate and hold this temperature. Use a small magnetic stir bar to mix the reactants at 350 R.P.M.for 5 minutes.
Make sure the BNC is appropriately impregnated with the ferrous solution and the reactants are completely dissolved. Increase the stirring velocity to 700 R.P.M.In a time interval of five minutes, add five milliliters of ammonium hydroxide to the 10 milliliters of ferrous solution using a pipetting needle that has been punched in a septum stopper. After addition of the ammonium hydroxide, the color of the solution changes from yellow-orange to black.
Continue stirring the solution at 80 degrees Celsius for another five minutes. Avoid high speed stirrings to maintain the integrity of the sample. Lower the temperature of the solution to 30 degrees Celsius using the temperature control bottom of the stirring hot plate and keep stirring for another five minutes.
Then, turn off the hot plate. At this point, the iron oxide nanoparticles have been incorporated into the BNC mesh. Next, cool the mixture down to room temperature and transfer it to a vessel flask to separate the magnetic nano-particles, or MNPs, and BNC with a strong permanent magnet.
To do this, keep the magnet close to the vessel to hold the MNPs and BNC in place while decanting the supernatant. Re-suspend the MNPs and BNC in 100 milliliters of water. Gently shake the solution to remove all the MNPs that are not strongly incorporated into the BNC.
Then, decant the supernatant again by holding the MNPs and the BNC in place using the magnet. Wash the MNPs and the BNC several times with water until the supernatant reaches neutral PH, as measured using a colorimetric strip. Separate the magnetic functionalized BNC, or MBNC, from the MNPs, using tweezers, and rinse the MBNCs several times with water until the water runs clear.
Sterilize the MBNC by exposing it overnight to U.V.Store the MBNC in 20 milliliters of deoxygenated high purity water that has been autoclaved at 120 degrees Celsius for 20 minutes. Aseptically, immerse the sample in one percent of polyethylene glycol, or PEG, and stir for two hours at room temperature. Coating with PEG improves the biocompatibility and stability of the iron oxide nano-particles deposited in the BNC as it is distributed over the MBNC three-D network.
The macroscopic appearance of the BNC is shown here. The culture vessel gives the shape of the pellicles. Scanning electron microscopy reveals that fine ribbons form the micro-structure of the BNC with approximately 50 nanometers in diameter, which create open pores across the entire network.
Magnetic functionalization of the BNC gives a less compacted fibril structure as compared to the BNC. The iron oxide nano-particles are preferentially located between the pores formed by fibril interlacing. Atomic force microscopy topography detected the magnetic force gradient characterized by two domains across the MBNC surface.
A high intensity magnetic field, shown in yellow, and a weak intensity magnetic field, shown in green. The presence of magnetic domains observed with MFM is confirmed by vibrating sample magnetometer data which shows that the iron oxide nano-particles are superparamagnetic, hence rendering the BNC magnetic. The integrity of the DNA on human aortic smooth muscle cells is preserved after a 24 hour incubation period, when cultured in the absence, and presence, of BNC or MBNC, as evident when compared to cells that underwent hydrogen peroxide treatment.
After watching this video, you should have a good understanding of how to render a bacterial nanocellulose magnetic for applications in damaged blood vessel reconstruction. We first had the idea for this method when we needed to induce a density gradient of magnetic nano-particles that had to be dispersed within the complex matrix of the bacterial nanocellulose network. Integrating the iron oxide nano-particles in this network required that we conducted a method during synthesis of the BNC.
Generally, individuals new to this method will struggle because there are a number of complex steps to impregnate iron oxide nano-particles into BNC.
