This work was funded by Department of Defense under contract No. W81XWH-11-2-0067

1. Preparation of Bacterial Nanocellulose (BNC)
Note: All of the steps are performed under aseptic conditions, unless otherwise indicated.
<ol>
	<li>Prepare culture medium.
	<ol>
		<li>Prepare 500 ml of liquid culture medium by combining 25 g of yeast extract, 15 g of peptone, 125.0 g of mannitol, and 500 ml of high purity water. Autoclave this mixture at 120 &#176;C for 20 min and store at 4 &#176;C.</li>
		<li>Prepare 100 ml of semisolid media by adding 15 g of agar to 5.0 g of yeast extra...

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The thickness and size of the BNC pellicle can be easily manipulated by changing the incubation time and the size of the flask in which it is grown during static cultivation. The microproperties of the BNC, such as porosity, can be modified by changing the oxygen ratio in the static culture. Higher oxygen concentrations yield tougher BNC11. A. Bodin and coworkers produced tubes of BNC with a burst pressure up to 880 mm Hg by changing the oxygen ratio from atmospheric oxygen to 100% oxygen during the fermentati...

The bacterian nanocellulose (BNC) is synthesized by Acetobacter xylinum strain, also known as Gluconacetobacter xylinus, and deposited in the form of films or pellicles on the air-liquid interface during stationary culture. These BNC pellicles adopt the form of the container where they are grown, and their thickness depends on the number of days in culture. A. xylinus uses the glucose in the medium for the synthesis of the cellulose microfibrils through a process of polymerization and subsequent crystallization. The polymerization of the glucose residues is carried out at the bacterial extracellular membrane where glucan chains are extruded from single pores distributed over the cell envelope. The crystallization of the cellulose microfibrils occurs in the extracellular space with the formation of glucan chain sheets by van der Waals bonding followed by stacking of the sheets by H-bonding1.
Magnetic nanoparticles integrated to a BNC matrix can be manipulated easily by an external magnetic field in order to increase the force necessary to direct and confine smooth muscle cells (SMCs) containing magnetic nanoparticles, at the damaged site of the arterial wall. This strategy keeps the SMCs away from other tissues, and holds the cells in place against the force exerted by the blood flow. It has been shown that SMCs play an important role in the vasoelasticity of the blood vessel, where they form abundant layers located mainly in the tunica media2.
The method used for the synthesis of MBNC involves BNC pellicle immersed and stirred in a solution of iron(III) chloride hexahydrate and iron(II) chloride tetrahydrate at 80 &#176;C. Ammonium hydroxide is added to form iron oxide nanoparticles inside the BNC mesh. The addition of ammonium hydroxide changes the color of the solution from orange to black. The IONPs compact together along the BNC fibrils with a non-uniform distribution.
This protocol focuses on the design of a bacterial nanocellulose-magnetic nanoparticle pellicle, which we have named magnetic bacterial nanocellulose (MBNC), which is intended to use as a substitute for missing, damaged or injured small-diameter blood vessels. H. S. Barud and coworkers have recently published a similar work to produce a BNC-based flexible magnetic paper by mixing BNC pellicles in a stable aqueous dispersion of PEG and superparamagnetic iron oxide nanoparticles3. Here, we describe the production of bacterial cellulose and its impregnation in situ with magnetic nanoparticles. A cytotoxicity assay based on detection of single DNA strand breaks was used to test the biocompatibility of the BNC and MBNC pellicles.

<table border="0" cellpadding="0" cellspacing="0" width="762">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Glucoacetobacter Xylinus</td>
			<td style="width:167px;">ATCC</td>
			<td style="width:101px;">700178</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Agar</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:101px;">A1296-500G&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">D-Mannitol Bioxtra</td>
			<td>Sigma Aldrich</td>
			<td style="width:101px;">M9546-250G&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Yeast Extract</td>
			<td style="width:167px;">BD Biosciences</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Bacteriological Peptone</td>
			<td>Sigma Aldrich</td>
			<td>P0556</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Sodium Hydroxide, 50% Solution In Water</td>
			<td>Sigma Aldrich</td>
			<td style="width:101px;">158127-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Iron(III) Chloride Hexahydrate</td>
			<td>Sigma Aldrich</td>
			<td style="width:101px;">236489-100G&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Ammonium Hydroxide&#160;</td>
			<td>Macron Fine Chemicals</td>
			<td style="width:101px;">6665-46</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Poly(Ethylene Glycol), Average Mn 400</td>
			<td>Sigma Aldrich</td>
			<td style="width:101px;">202398-250G&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Iron (II) chloride tetrahydrate</td>
			<td>Sigma Aldrich</td>
			<td style="width:101px;">44939-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Disposable petri dish</td>
			<td>Sigma Aldrich</td>
			<td>BR452000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Disposable Inoculating Loop</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td>22-363-604&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Anhydrous Calcium Sulfate</td>
			<td style="width:167px;">W.A. Hammond Drierite&#160;</td>
			<td style="width:101px;">13001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">High vacuum grease</td>
			<td>Sigma Aldrich</td>
			<td>Z273554-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Laboratory pipetting needle with 90&#176; blunt ends</td>
			<td>Sigma Aldrich</td>
			<td>CAD7937-12EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">pH test strips&#160;&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P4786-100EA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Round-bottom three neck angle type distilling flask</td>
			<td>Sigma-Aldrich</td>
			<td>CLS4965250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Silicone oil for oil baths</td>
			<td>Sigma-Aldrich</td>
			<td style="width:101px;">85409-250ML&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Drying Tube</td>
			<td style="width:167px;">Chemglass</td>
			<td>CG-1295-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Septum Stopper, Sleeve Type</td>
			<td style="width:167px;">Chemglass</td>
			<td>CG-3022-98</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Magnetic stir bar</td>
			<td style="width:167px;">Chemglass</td>
			<td>CG-2001-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Condenser</td>
			<td style="width:167px;">Chemglass</td>
			<td>CG-1218-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Temperature Controller</td>
			<td style="width:167px;">BriskHeat</td>
			<td>SDC120JC-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Stirring Hotplate</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td>11-100-49SH&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Comet Assay Kit</td>
			<td style="width:167px;">Trevigen</td>
			<td>4250-050-K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">SYBR Gold Nucleic Acid Gel Stain</td>
			<td style="width:167px;">Life Technologies</td>
			<td>S-11494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">bio-AFM</td>
			<td style="width:167px;">JPK Instruments</td>
			<td colspan="2">NanoWizard 4a BioScience AFM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Nanoindenter</td>
			<td style="width:167px;">Micro Materials Ltd</td>
			<td colspan="2">Multi-module mechanical tester&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Scanning electron microscopy (SEM)</td>
			<td>Hitachi High Technologies America</td>
			<td>Hitachi S-4800</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of a functionalized magnetic bacterial nanocellulose with iron oxide nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

The incubation period of G. xylinus was a total of 9 days, but the pellicles began to form earlier and were evident after about 2 days. The macroscopic appearance of the BNC is displayed in Figure 1, the shape of which mimics that of the dish-grown culture. Figure 2 describes the process for producing BNC-IONP pellicles, which summaries the main steps involved in the protocol above as well as the configuration of the main compone...

Watch this Scientific Journal Video about Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles at JoVE.com

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

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.

fabrication-functionalized-magnetic-bacterial-nanocellulose-with-iron

Research

JoVE Journal

Bioengineering

12.9K vistas.  University of Illinois at Urbana-Champaign. 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 vasc...