(72b) Direct Control of Biological Assembly Using Electrokinetic Forces

Introduction

Existing scaffold fabrication techniques for tissue engineering suffer from the distinct lack of processes capable of reproducibly creating structures on the nano-, micro-, and millimeter scales that adequately promote cell growth and function. We have made steps towards overcoming this limitation by manipulating the shape and nanoscale morphology of bacterial cellulose material. Cellulose, a natural polymer produced by the majority of plants, can be assembled into nanofibrils by bacteria. This bacterial cellulose has many attractive properties as a scaffolding material on which to seed cells for regenerative medicine including biocompatibility, mechanical integrity, hydroexpansivity, and stability under a wide range of conditions [1].

Figure 1: The dense random cellulose network produced naturally by A. xylinum.

The bacteria Acetobacter xylinum produces nanoscale cellulose ribbons at an average rate of 2microns/min [2] creating a random cellulose network. In order for cellulose networks to be viable solutions for biomedical applications, such as implants and tissue scaffolds, their orientation must be controlled [3] to ?remind? cells of the nanotopography of their natural extracellular matrix. We hypothesize and demonstrate that small electric fields can be used to electrokinetically direct these cells as they produce nanocellulose thus providing a means to control the orientation of these fibers.

Figure 2: a) Fabrication process for creating microchannels. b) Schematic showing the top view of the channels with insulating pillars. c) Experimental setup for interfacing with microfluidic channels. d) Fluorescently labeled A. xylinum cells being controlled through a microchannel dielectrophoretic forces.

Dielectrophoresis (DEP) is the motion of a particle due to its polarization induced by the presence of a non-uniform electric field [4]. The velocity of the bacteria under an electric field is calculated by:

where µ_ek and µ_DEP are intrinsic parameters of the bacteria in their culture media. DEP has been shown is an effective means to manipulate and differentiate cells based on their size, shape, internal structure, and intrinsic properties such as conductivity and polarizability [5]. Insulator-based dielectrophoresis (iDEP) uses insulating obstacles, instead of electrodes, within the device to produce spatial nonuniformities in the electric field [6-8].

Methods

We have used the strain Acetobacter xylinum subspecies sucrofermentas BPR2001, trade number 700178?, from the American Type Culture Collection. They were cultured in a modified fructose media with an addition of corn steep liquid. For precultivation, 6 cellulose-forming colonies were cultured for 2 days at 30ºC in a 300ml rough flask. The bacteria will be liberated by vigorous shaking and inoculating in the desired amount into the culture media.

To study the response of A. xylinum to such electric fields, we created microfluidic devices in PDMS using a silicon master stamp fabricated using standard photolithography and deep reactive ion etching. A typical schematic of one of our devices is presented in Figure 2b. To cultivate samples large enough for mechanical testing and cell seeding experiments, larger 500 micron deep micro-chambers were fabricated using silicon stamps.  A. xylinum cells in culture media were injected into the microfluidic channels and pressure was allowed to equalize. Platinum electrodes were then used to apply small electric fields across the channels inducing electrokinetic and dielectrophoretic forces that guided the bacterial cells as they produced cellulose nanofibers.

Results  

Experimental parameters including the strength of the applied field, chamber dimensions, and cultivation time have each been individually examined to determine the optimal parameters for electrokinetic control of this biofabrication process. In small geometries, the cellulose produced quickly clogs the channels and electrokinetic movement of the bacteria is halted. In large geometries such as cuvettes and test tubes, electromagnetic forces no longer dominate. If the applied electric field is too low, the dielectric forces fail to navigate the bacteria through the microfluidic environment. In these scenarios a random cellulose network is produced. Conversely, if the applied field is strong enough for dielectrophoretic forces to become dominant, the bacteria are moved too quickly or thermally strained and cellulose producing mechanism within the bacteria is switched off.

We have found that we can control the morphology of the cellulose scaffolds when the bacteria are subjected to electric fields between 0.25V/cm and 1.0V/cm, are in a 19 mm long, 500 micron deep PDMS encased microfluidic chamber, and allowed to produce cellulose for 48 hours while being guided through the chamber by electrokinetic forces. At these field strengths, the bacterial cells are being controlled with velocities on the order of 1 micron/s.

Figure 3: FESEM of the cellulose network produced by A. xylinum under an applied electric field.

A FESEM image of the cellulose produced under 0.45V/cm in which interwoven strands of nanocellulose fibrils are aligned in the direction of the applied electrical fields is shown in Figure 3. Variations in the strength of the applied field change the morphology of the cellulose structure produced. Figure 3 is in clear contrast with Figure 1 which shows a randomly distributed network of nanocellulose. The ellipsoid shaped particles on top of the strands in Figure 3 are the bacteria which have been fixed to the cellulose fibers during the freezing process.  The results in the Figure 3 clearly show that the orientation of cellulose fibers and the architecture of the network can be predictably controlled using electric fields.

Conclusion

This process can be adapted to mimic the complex fiber orientations found in physiological tissues. The ability to control the direction of fiber orientation could be readily expanded to weave structures of multiple fiber layers, with each layer grown in a prescribed direction, by simply changing the orientation of the applied electric field and these structures could be tailored to have mechanical properties that match those of the extracellular matrix of targeted tissues.

Future work will focus on the creation of scaffolds for tissue engineering which will require adapting this technique on much larger scales in which Joule heating may become lethal to the bacterium. The use of varying geometries to increase the dielectrophoretic force applied while keeping the applied field low or use emerging technologies such as contactless dielectrophoresis [9] are currently under investigation.

Acknowledgements

We acknowledge the Institute for Critical Technologies and Applied Science (ICTAS) at Virginia Tech for financial support and the NCFL at ICTAS for support with imaging. We thank Dr. Aase Bodin for introducing Mike Sano to the bacterial cellulose field as well Hadi Shafiee for experimental assistance.

References

1.     Helenius, G., et al., In vivo biocompatibility of bacterial cellulose. Journal of Biomedical Materials Research Part A, 2006. 76A(2): p. 431-438.

2.     Brown, R.M., J.H.M. Willison, and C.L. Richardson, Cellulose Biosynthesis In Acetobacter-Xylinum - Visualization Of Site Of Synthesis And Direct Measurement Of Invivo Process. . Proceedings of the National Academy of Sciences of the United States of America, 1976. 73(12): p. 4565-4569.

3.     Griffith, L.G. and G. Naughton, Tissue engineering - Current challenges and expanding opportunities. Science, 2002. 295(5557): p. 1009-+.

4.     Pohl, H.A., The Motion and Precipitation of Suspensoids in Divergent Electric Fields. Journal of Applied Physics, 1951. 22(7): p. 869-871.

5.     Gascoyne, P.R.C., et al., Dielectrophoresis-based programmable fluidic processors. Lab on a Chip, 2004. 4(4): p. 299-309.

6.     Lapizco-Encinas, B.H., et al., An insulator-based (electrodeless) dielectrophoretic concentrator for microbes in water. Journal of Microbiological Methods, 2005. 62(3): p. 317-326.

7.     Davalos, R.V., et al., Performance impact of dynamic surface coatings on polymeric insulator-based dielectrophoretic particle separators. Analytical and Bioanalytical Chemistry, 2008. 390(3): p. 847-855.

8.     Martinez-Lopez, J.I., et al., Characterization of electrokinetic mobility of microparticles in order to improve dielectrophoretic concentration. Analytical and Bioanalytical Chemistry, 2009. 394(1): p. 293-302.

9.     Shafiee, H., et al., Contactless Dielectrophoresis: A New Technique For Cell Manipulation. Biomedical Microdevices, 2009.