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Description
Our team came up with the idea of a decentralized source of electric stimulation instead of plugging or using batteries for tissue engineering. The preliminary research showed that fish-scale is rich in type I collagen which is a biocompatible and biodegradable polymer suitable for building the flexible bio-piezoelectric nanogenerator [1]. This can be taken as temporal electric signals which are to be used for the tissue engineering scaffold. Hence we examine different kinds of movements to see if its induced electric potential can be used for the scaffold.
In this section, we tested induced electric potential with a varying treatment duration of demineralization by ethylenediaminetetraacetic acid (EDTA). The freshwater fish scales are soaked in EDTA for 0h, 2h, 4h, 8h, 16h, and 24h. Either 1 or 4 fish scales are fabricated with polypropylene (PP) film with a voltmeter to record the voltage during tapping rigorously. The average voltage generated by the piezoelectrical effect of fish-scale, Uavg, is calculated by Equation 1.
where Uimax and Uimin is the intrinsic potential without tapping.
Our team came up with the idea of a decentralized source of electric stimulation instead of plugging or using batteries for tissue engineering. The preliminary research showed that fish-scale is rich in type I collagen which is a biocompatible and biodegradable polymer suitable for building the flexible bio-piezoelectric nanogenerator [1]. This can be taken as temporal electric signals which are to be used for the tissue engineering scaffold. Hence we examine different kinds of movements to see if its induced electric potential can be used for the scaffold.
In this section, we tested induced electric potential with a varying treatment duration of demineralization by ethylenediaminetetraacetic acid (EDTA). The freshwater fish scales are soaked in EDTA for 0h, 2h, 4h, 8h, 16h, and 24h. Either 1 or 4 fish scales are fabricated with polypropylene (PP) film with a voltmeter to record the voltage during tapping rigorously. The average voltage generated by the piezoelectrical effect of fish-scale, Uavg, is calculated by Equation 1.
U avg = 1⁄2 (| U max - U imax| + | U min - U imin|) (1)
where Uimax and Uimin is the intrinsic potential without tapping.
Results
The results are shown in Table 1 and Figure 1, and the sample response in Fig 2 (8 hours & 4 FSs).
Table 1: Recorded voltages of each group of fish-scale
Fig 1: Voltage-Generated by FSCs with different EDTA treatment duration
Fig 2: 4 Fish Scales with 4 hours EDTA responding to tapping
Fig 3: Setup for Fish Scale Voltage Measurement
From our experiment, it can be concluded that the piezoelectrical effect of fish-scales increases as the EDTA treatment time increases and larger voltages can be generated by putting more fish-scales together. Especially, the batch of fish scales (4 FSC) significantly increased its potential after 24 hours of demineralization.
The results are shown in Table 1 and Figure 1, and the sample response in Fig 2 (8 hours & 4 FSs).
| EDTA [h] | #FSC | Uimax[mV] | Uimin[mV] | Umax[mV] | Umin[mV] | Uavg[mV] |
|---|---|---|---|---|---|---|
| 0 | 1 | -3.29 | -4.7 | -2.35 | -6.11 | 1.175 |
| 0 | 4 | 1.88 | 0 | 3.29 | -1.41 | 1.41 |
| 2 | 1 | -1.41 | -3.29 | 0 | -5.64 | 1.88 |
| 2 | 4 | 0.94 | -0.94 | 2.82 | -5.71 | 3.325 |
| 4 | 1 | 1.41 | 0 | 3.29 | -1.88 | 1.88 |
| 4 | 4 | 1.41 | -0.47 | 8.46 | -5.64 | 6.11 |
| 8 | 1 | 1.41 | -0.94 | 2.82 | -2.35 | 1.41 |
| 8 | 4 | 0.94 | -0.94 | 6.11 | -3.76 | 3.995 |
| 16 | 1 | 1.88 | -0.47 | 3.29 | -3.29 | 2.115 |
| 16 | 4 | 1.41 | 0 | 7.52 | -6.58 | 6.345 |
| 24 | 1 | 0.94 | -0.94 | 3.29 | -3.76 | 2.585 |
| 24 | 4 | 2.82 | 0 | 62.51 | -44.18 | 51.935 |
From our experiment, it can be concluded that the piezoelectrical effect of fish-scales increases as the EDTA treatment time increases and larger voltages can be generated by putting more fish-scales together. Especially, the batch of fish scales (4 FSC) significantly increased its potential after 24 hours of demineralization.
Summary
None of the sets was producing enough electric potential to control the electrochemical modulators used later in our projects (~0.5 V [2]). However, there are several improvements that could be made. First, since we tapped the fish scales by hand, the force of each tapping was varying. Second, sometimes we accidentally tapped on the wires instead of the fish scales, which would generate extremely large voltages. Third, more different types of fish-scales can be used such as seawater fish. For future experiments, we can further investigate more experimental groups with the EDTA treatment time ranging from 16hours to 48 hours. Also, not only tapping but also bending and other possible mechanical stress that could be made for the implanted scaffolds.
None of the sets was producing enough electric potential to control the electrochemical modulators used later in our projects (~0.5 V [2]). However, there are several improvements that could be made. First, since we tapped the fish scales by hand, the force of each tapping was varying. Second, sometimes we accidentally tapped on the wires instead of the fish scales, which would generate extremely large voltages. Third, more different types of fish-scales can be used such as seawater fish. For future experiments, we can further investigate more experimental groups with the EDTA treatment time ranging from 16hours to 48 hours. Also, not only tapping but also bending and other possible mechanical stress that could be made for the implanted scaffolds.
Description
The next step is to establish our own method of electrochemical modulation to prove our tissue engineering model is possible. we use two redox biomolecules: pyocyanin (Pyo) for the initiation of gene induction and ferricyanide (Fcn) for electronic control of the expressions [2]. The existing part, BBa_K2862021, is used for our initial step. Agar plates are dug by the carbon electrode that gives +0.5V. (Fig 4). The GFP fluorescence level is determined by pixel intensities after a 1-hour electric shock followed by 24-hour post-culturing.
Fig 4: Electric Modulation on Agar Plate Setup
The next step is to establish our own method of electrochemical modulation to prove our tissue engineering model is possible. we use two redox biomolecules: pyocyanin (Pyo) for the initiation of gene induction and ferricyanide (Fcn) for electronic control of the expressions [2]. The existing part, BBa_K2862021, is used for our initial step. Agar plates are dug by the carbon electrode that gives +0.5V. (Fig 4). The GFP fluorescence level is determined by pixel intensities after a 1-hour electric shock followed by 24-hour post-culturing.
Results
The sample with electricity and pyocyanin (+E/+Pyo) showed the biggest intensity, and the sample without electricity but still with pyocyanin (-E/+Pyo) showed a modest intensity. This clearly proved that our electric signals enhanced the expression level on the existing part (BBa_K2862021).
Fig 5: Results of PixCell Construct
The sample with electricity and pyocyanin (+E/+Pyo) showed the biggest intensity, and the sample without electricity but still with pyocyanin (-E/+Pyo) showed a modest intensity. This clearly proved that our electric signals enhanced the expression level on the existing part (BBa_K2862021).
Summary
As the result is shown in Fig 5, the electric stimulation properly worked on the existing parts in a much simpler method with a normal power supply than the previous method with an expensive potentiostat. This result characterizes the existing part (BBa_K2862021) and improves it since we introduced a much simpler method of electric activation and created the reporter gene of more complicated pathways regulated by electric stimulation, which serves as one of our gold medal criteria.
As the result is shown in Fig 5, the electric stimulation properly worked on the existing parts in a much simpler method with a normal power supply than the previous method with an expensive potentiostat. This result characterizes the existing part (BBa_K2862021) and improves it since we introduced a much simpler method of electric activation and created the reporter gene of more complicated pathways regulated by electric stimulation, which serves as one of our gold medal criteria.
Description
Finally, we exhibited something novel by using the electrochemical control of genes and proved that complex signaling is possible by our piezoelectric scaffolding for future tissue engineering. We used Quorum-Sensing genes as our model (fig 6) and tested whether electric stimulation can flow down to the GFP expression. Two parts (Relay cell and Receiver cell) are separately transformed in E. coli. and mixed on the agar plate to cultured under electric stimulation. Similar to Part 2, the GFP fluorescence level is determined by pixel intensities after a 1-hour electric shock followed by 24-hour post-culturing. There are three sets to show our model in Fig 5: Co-culture of both cells, only relay cell, and only receiver cell.
Finally, we exhibited something novel by using the electrochemical control of genes and proved that complex signaling is possible by our piezoelectric scaffolding for future tissue engineering. We used Quorum-Sensing genes as our model (fig 6) and tested whether electric stimulation can flow down to the GFP expression. Two parts (Relay cell and Receiver cell) are separately transformed in E. coli. and mixed on the agar plate to cultured under electric stimulation. Similar to Part 2, the GFP fluorescence level is determined by pixel intensities after a 1-hour electric shock followed by 24-hour post-culturing. There are three sets to show our model in Fig 5: Co-culture of both cells, only relay cell, and only receiver cell.
Results
The quantitative result shows the straightforward outcome (Fig 7): only when both Relay and Receiver cell exists (Co-culture), GFP expression is enhanced. Only the Receiver cell has lightly more expression of GFP than only Relay cell. This should be due to the intrinsic LuxR proteins that facilitate the GFP expression only by its own plasmid. Qualitatively, we also observed the spatial expressing patterns of quorum sensing as most of the GFP expressing cells are located as if the glowing colonies are surrounding the positive electrode on the left side (Fig 8). This is because AHL molecules diffused from the Relay cells around the positive electrode and Receiver cells responded to the gradient of Quorum-sensing inducers.
Fig 7: Results of Quorum Sensing Cells
Co-culture Plate under GFP (the left hole is positive anode; the right hole is the negative cathode)
The quantitative result shows the straightforward outcome (Fig 7): only when both Relay and Receiver cell exists (Co-culture), GFP expression is enhanced. Only the Receiver cell has lightly more expression of GFP than only Relay cell. This should be due to the intrinsic LuxR proteins that facilitate the GFP expression only by its own plasmid. Qualitatively, we also observed the spatial expressing patterns of quorum sensing as most of the GFP expressing cells are located as if the glowing colonies are surrounding the positive electrode on the left side (Fig 8). This is because AHL molecules diffused from the Relay cells around the positive electrode and Receiver cells responded to the gradient of Quorum-sensing inducers.
Summary
Overall, our parts are proved to be working under the electric stimuli as it is showing the GFP expression with the spatial properties of quorum sensing. The quantitative analysis by pixel intensities is conducted very carefully (using the exact same spot for every picture) to minimize the other factors. Therefore, we combined with qualitative analysis of a single plate to reinforce our results.
Overall, our parts are proved to be working under the electric stimuli as it is showing the GFP expression with the spatial properties of quorum sensing. The quantitative analysis by pixel intensities is conducted very carefully (using the exact same spot for every picture) to minimize the other factors. Therefore, we combined with qualitative analysis of a single plate to reinforce our results.
References
[1] Ghosh, S. K. & Mandal, D. High-performance bio-piezoelectric nanogenerator made with fish scale. Appl. Phys. Lett. 109, 103701 (2016).
[2] 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
[1] Ghosh, S. K. & Mandal, D. High-performance bio-piezoelectric nanogenerator made with fish scale. Appl. Phys. Lett. 109, 103701 (2016).
[2] 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