To develop a novel piezoelectric platform to probe mechanobiological interactions. This pilot project serves to validate the basic process and has several key objectives. The first goal is to successfully grow a viable cell colony on the piezoelectric matrix. If that can be achieved, then we can determine whether or not the traction forces exerted by the cell culture can be detected and monitored as the culture grows.
The Idea
Mechanobiology is an emergent field of research concerned with the mechanical interactions between biological systems at a cellular level. It is becoming increasingly evident that the mechanical environment of a cell is crucial in determining its behaviour, and the subsequent long range morphology of multicellular systems [1], [2]. This proposal aims to probe these interactions using a novel piezoelectric platform.
If the mechanisms behind mechanical interactions can be determined, then the possibility exists to manipulate the function of cells by precisely controlling the biophysical forces they experience. The development of a platform that allowed manipulation of biophysical forces independently from the metabolic and signalling pathways engineered inside a cell would be a powerful aid in the development of synthetic biology.
Perhaps the most exciting possibility of such an advance would be dynamic remodelling of tissues in a manner analogous to bone remodelling. External and addressable control over intercellular mechanical signals could have numerous medical or industrial applications for generating advanced composite materials in arbitrary shapes using low intensity energy sources, and would represent an entirely new route to fabrication of bulk materials. Such mechanical signals are critical in morphogenesis and the details of their effects are poorly understood, although general mechanisms are known. For example, cells can physically interact with each other and their surrounding surfaces via membrane bound adhesins which can detect and anchor on to specific external sites, or membrane bound integrins can dock with extra cellular matrix (ECM) such as collagen or other integrins and transduce mechanical signals across the membrane into the cell. In response to such external stimuli, various effects can arise such as the production of ECM (curli fibres in E.coli or collagen in eukaryotic cells), which results in further signalling for nearby cells. Such feedback loops lead to important cues for cells, particularly in multicellular systems where they can trigger differentiation and tissue remodelling processes. With the platform outlined in this proposal, the possibility arises for directly sensing and actuating extracellular forces within a cellular environment under external and addressable dynamic control.
Various techniques have been developed to observe the forces involved in cellular interactions, and all have their advantages and disadvantages [3], [4] . For example, Atomic Force Microscopy (AFM) is sensitive to extremely small forces (of the order of piconewtons) and offers very fine spatial resolution, but at any one time it is only possible to probe a single point. The use of optical tweezers allows for manipulation of a handle placed on the surface of a cell, yet this requires complex equipment. The current techniques all require specialist equipment and are time consuming. We would like to develop a simple and inexpensive test bed that would allow for a high throughput of synthetic biology experiments with minimal unwanted influence on the cell behaviour.
We believe that nanostructured piezoelectric materials are well suited to creating such a device. Cells are typically micron sized and exert forces in the pico- to nanonewton range. By creating piezoelectric structures of similar dimensions, these forces can be transduced since piezoelectric materials become polarised when deformed. By allowing a culture of cells to act on and deform a piezoelectric structure, the resulting polarisations could be detected using an array of electrodes and used to image the interactions occurring.
Furthermore, the same imaging platform could also be used to stimulate the growing cells. As the piezoelectric effect also works in reverse, applying an electric field will induce a deformation of the material. An array of microscopic pre-patterned electrodes will allow precise spatial control of this deformation.
Who we are
Implementation
The aim of this proposal is to explore the scope of using piezoelectric materials to sense and stimulate the function of cells. This project will be a feasibility study to ensure that cells can be cultured on such a device and to determine if the forces that they exert can be detected though the piezoelectric effect.
The rapid prototyping of test bed devices will be carried out using a state-of-the-art Optomec Aerosol Jet printer, recently installed in the Materials Science department. This allows for large area deposition of micron-scaled features and will be a valuable tool for testing various device designs and materials.
The piezoelectric material used in these devices will require some investigation, but will be focussed on piezoelectric polymers. Poly(vinylidene fluoride) (PVDF) is a well characterised piezoelectric polymer that is bio-compatible. It has been the subject of recent investigation for energy harvesting purposes and can easily be fabricated into nanowires [5]. Polylactic acid (PLA) is another piezoelectric polymer which is also biodegradable. However, the properties of this polymer are not well understood. Research into PLA will continue in parallel with this project and may inform materials selection. Electrode material and design will also be investigated.
Throughout the production process, the materials used will need to be thoroughly characterised.
This will involve use of techniques such as Scanning Electron Microscopy (SEM), Piezo-Force Microscopy (PFM) and Differential Scanning Calorimetry (DSC). Once a satisfactory device has been manufactured and characterised, work will focus on encouraging cells to grow on the platform.
Initially, E.coli cells will be used for this investigation due in part to their immediate availability and
their robustness but also because of the possibility of engineering their external-curli fibres should the sensor platform prove viable.
This pilot project serves to validate the basic process and has several key objectives. The first goal is to successfully grow a viable cell colony on the piezoelectric matrix. If that can be achieved, then we can determine whether or not the traction forces exerted by the cell culture can be detected and monitored as the culture grows. If both of these objectives can be attained, then we will be able to perform targeted mechanical stimulation of the cells and observe the effect. Following this, a number of possible avenues could be explored including engineering ECM or introducing more specialised or complex cells, such as Eukaryotic cells, to investigate the role of piezeo-electric effects in bone formation.
Benefits and outcomes
This work will represent novel use of piezoelectric materials within the realm of biology.
Piezoelectric biosensors do exist, but these operate on a very different principle. This approach to biosensing has not yet been explored, partly due to the fact a method of creating nanostructures from bio-compatible piezoelectric material has only recently been developed [5].
The idea behind this project exists at the interface between several disciplines including biology, physics and materials science. As such, interdisciplinary work is necessary to ensure a sufficient knowledge base. Support from the SynBio fund will allow this collaboration, bringing new perspective to all aspects of the project. Biochemists will bring experience with handling biological samples and of cell behaviour whilst the device design and manufacture can be determined within materials science. This project is targeted at producing a tool that will facilitate synthetic biology through the manipulation of the mechanical environment of cells. The development of the platform will be openly documented and we hope to publish any discoveries that result from its use in an open access paper. Once we have established the basic viability of such a tool it's development will proceed far more quickly in an open source environment, particularly because our approach is so simple. This project will focus within the realm of synthetic biology, however the applications of this technology could well extend beyond. Future work may investigate the use of a similar technique to produce low cost medical sensors.
Budget
£4000 initially, plus £1000 at end of 6 months if goals are achieved.
Publishing open access paper – from £1000 follow-on grant
Consumables for printer: inks, substrates, specialist cleaning equipment for use with biological materials, dedicated labware - £1200
Facilities charges: Scanning Electron Microscopy (SEM), Differential Scanning Calorimetry (DSC), Infrared spectroscopy (IR), X-Ray diffraction (XRD), Atomic Force Microscopy (AFM) for characterising the materials within the device - £1600
Biological reagents and wet lab consumables - £1200.
References
[1] B. D. Hoffman and J. C. Crocker, “Cell Mechanics: Dissecting the Physical Responses of Cells to Force,” Annu. Rev. Biomed. Eng., vol. 11, no. 1, pp. 259–288, 2009.
[2] V. Vogel and M. Sheetz, “Local force and geometry sensing regulate cell functions.,” Nat. Rev. Mol. Cell Biol., vol. 7, no. 4, pp. 265–275, 2006.
[3] E. Moeendarbary and A. R. Harris, “Cell mechanics: principles, practices, and prospects,” WIREs Syst Biol Med, vol. 6, pp. 371–388, 2014.
[4] D.-H. Kim, P. K. Wong, J. Park, A. Levchenko, and Y. Sun, “Microengineered Platforms for Cell Mechanobiology,” Annu. Rev. Biomed. Eng, vol. 11, pp. 203–33, 2009.
[5] R. a. Whiter, V. Narayan, and S. Kar-Narayan, “A Scalable Nanogenerator Based on Self-Poled Piezoelectric Polymer Nanowires with High Energy Conversion Efficiency,” Adv. Energy Mater., Aug. 2014.