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Tissue engineering requires three components: stem cells, growth and differentiation factors, and biomimetic scaffolding materials. Tissue engineering scaffolds are usually supporting cell structures, but the novel paradigm requires possible biomaterials in order to properly regenerate specific tissues without chemicals [1]. Particularly, the electric and electrochemical cues are among the most relevant ones in various methods of determining tissue functionality. The electroactive and biocompatible material, collagen, is found to be a great material for the novel therapeutic strategies because they are profound in human bones, muscle, and other human body parts [2, 3].

Fig 1: Typical Tissue Engineering Scheme[1]

The artificially produced collagens that mimic existing bone structures can be one possible way to discover, however, the natural collagen source has become our interest because our lab is not capable of constructing sophisticated biomaterials from scratch. We found fish scales could be used for the collagen source to create a tissue engineering scaffold. Previous researches [6, 7] showed that the fish scales have piezoelectricity, which means that they are capable of producing electric potential due to mechanical stress and vice versa. We aim at showing that the electric signal produced by the mechanical stress on the fish scale is able to control gene expressions of E. coli. for the novel therapeutic application of biowaste.

Collagen fibril is a plentiful protein in our body, which is made up of triple helix structures. Each helix contains the specific residue of Gly, (Ala,) Pro, and Hyp. Proline and Hydroxyproline smoothly bend the chain around the helix. As figure 2 has the red dot sphere, the hydroxyl group of Hyp contributes to the intrinsic presence of polar uniaxial orientation of hydrogen bonding between these polypeptide chains. Those chains act as molecular dipoles to harvest electricity.
Type I Collagen is biocompatible and biodegradable polymer enabling flexible scaffolds. The previous research showed that the freshwater fish scale does have the piezoelectricity but did not specify the necessary treatment clearly. We hypothesized that the demineralization step by EDTA is critical for a better piezoelectric potential generation. We quantities the piezoelectric potential by tapping for various EDTA treatment time of the grass fish scale (Ctenopharyngodon idella). Read more about our results here

Fig 2: Structure of Helical Tropocollagen [8]

Fig 3: Piezoelectric Nanogenerator Production Scheme [7]

We designed two parts to demonstrate that the complex cellular expression can be controlled by the electric potential. Fig 4 shows the overall schematic of our design. We have two parts in this experiment and transformed in the separate cell lines:

Bio-electric Relay part: BBa_K326700(For the Silver Medal criteria)
Quorum-Sensing Receiver part: BBa_K326701(For the Gold Medal criteria)

The Relay part has pSoxR/pSoxS bidirectional promoters. The redox-active molecule, Pyocyanin, initiates the expression of this part by activating the intrinsic SoxR transcription factor. Under the positive electric potential, reduced Ferrocyanide gets oxidized to become Ferricyanide, and signal is transferred to the intracellular environment via Menaquinone (MQ) is oxidized by Ferricyanide through the membrane. Then, the reduced Pyocyanin that initiated the reaction once again oxidized to activate SoxR protein. This positive feedback loop creates LuxI protein, which synthases the quorum sensing hormone, acyl-homoserine lactone (AHL).
The Receiver part receives the AHL signal when AHL density is high (Fig 5). AHL activates the transcription factor LuxR which binds to pLuxR/pLuxI promoter. LuxI coding sequence is replaced by a GFP coding sequence so that this part reports the signaling by fluorescence.
Those redox modulators are added in the agar, and cells are cultured with electric stimuli by the normal power supply. Then, cell-to-cell communication is demonstrated by the electric potential in the discussed manner. Read more about our results here

Fig 4: Overall Schematics of Electric Control Experiment]

Fig 5: Quorum Sensing Mechanism by AHL [9]

Tapping fish scales is not very appropriate because the cells are cultured on the plate. Instead, the soundwave is the best mechanical stimuli to manipulate our parts. The fish scales are fabricated with agar plates with necessary electrochemical modulators. Theoretically, the soundwaves with enough intensities will vibrate the plate and fish scales. Consequently, the piezoelectric potential will have the same effect as the power supply.

References

[1] C. Ribeiro, V. Sencadas, D.M. Correia, S. Lanceros-méndez. Piezoelectric polymers as biomaterials for tissue engineering applications.Colloids Surfaces B: Biointerfaces, 136 (2015), pp. 46-55

[2] Parenteau-Bareil, R., Gauvin, R., & Berthod, F. (2010). Collagen-Based Biomaterials for Tissue Engineering Applications.Materials, 3(3), 1863–1887. doi:10.3390/ma3031863.

[3] Jacob J., More N., Kalia K., Kapusetti G. Piezoelectric smart biomaterials for bone and cartilage tissue engineering. Inflamm. Regen. 2018;38:2. doi: 10.1186/s41232-018-0059-8.

[4] J.K. Wang, K.P. Yeo, Y.Y. Chun, T.T.Y. Tan, N.S. Tan, V. Angeli, C. Choong.Fish scale-derived collagen patch promotes growth of blood and lymphatic vessels in vivo. Acta Biomater., 63 (2017), pp. 246-260,10.1016/j.actbio.2017.09.001.

[5] T. Tschirhart, E. Kim, R. McKay, H. Ueda, H.C. Wu, A.E. Pottash, A. Zargar, A. Negrete, J.Shiloach, G.F. Payne, W.E. Bentley.Electronic control of gene expression and cell behaviour in Escherichia coli through redox signalling. Nat Commun, 8 (2017), p. 14030

[6] Ghosh, S. K. & Mandal, D. High-performance bio-piezoelectric nanogenerator made with fish scale. Appl. Phys. Lett. 109, 103701 (2016).

[7] Ghosh, S. K.; Mandal, D. Sustainable Energy Generation from Piezoelectric Biomaterial for Noninvasive Physiological Signal Monitoring. ACS Sustainable Chem. Eng. 2017, 5, 8836– 8843, DOI: 10.1021/acssuschemeng.7b01617

[8] Image of 1bkv: Kramer, R.Z., Bella, J., Mayville, P., Brodsky, B., Berman, H.M. (1999) Sequence-dependent conformational variations of collagen triple-helical structure. Nature Structural and Molecular Biology 6: 454-457.

[9] Li Z, Nair SK. Quorum sensing: how bacteria can coordinate activity and synchronize their response to external signals. Protein Sci 2012; 21(10): 1403–1417.