Electric and Magnetic Field Devices for Stimulation of Biological Tissues

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Biophysical stimulus have been used to stimulate the cell and molecular dynamics in different tissues. Certain studies have evaluated the impact of electric and magnetic fields in different kind of cells such as chondrocytes, osteoblasts and fibroblasts, tissue implants and scaffolds. Although different stimulatory devices have been developed under the specific features to stimulate biological tissues, it is necessary to confirm electric and magnetic devices in which the voltage and frequency could be varied to stimulate a wide range of biological samples.

The computational simulation to verify the electric field distribution was performed in COMSOL Multiphysics. Here, an axisymetric configuration was used to simulate the capacitive system which is composed by two parallel electrodes, the air, our culture well plate, the culture media, and the biological sample, that in this case, has been represented by a scaffold. The material properties of each element were the electric conductivity and the relative permittivity.

The applied voltage was set at 100 volts, while the frequency was set at 60 kilohertz sine Wien for. After all parameters were introduced, the model is calculated to observe the electric field distribution in all surfaces. To observe in a more detailed way the electric field distribution, it is possible to plug the electric field in the complete system, within the scaffold, in the culture media, in the air, and within the cultural well plate and outside the electrodes.

The circuit will generate the electric fields, is based on the bridge wien oscillator. This is a RCnonthian sequence of face chip which uses both positive and negative feedback. The bridge wien oscillator is composed by a lit lab network that is a reactive voltage divided in which the input voltage is divided by the combination between R5 and C2 in series and by the combination between R6 and C3 in parallel.

To calculate the frequency, we use a resonant frequency equation where F Sub-Zero is the frequency R equal to R5 equal to R6 are the resistors and C equal to C2 and C3 are the capacitors. This circuit is designed so that the resistive voltage divided increases when the output that increases in amplitude and the resistive voltage divided decreases when they output voltage decreases in amplitude. Thus, the voltage gain of the amplifier commences automatically the amplitude changes of the output signal.

Then a combination of resistors was calculated to generate the four upper voltages. Finally, a signal rectification stage was implemented before to amplify the signal with the transformer. Once the circuit was simulated, the painted circuit board was manufactured as a result the final sinusoidal signal generated by the oscillator is plot.

Once the circuit is simulated the next step is to build the wien bridge oscillator in a breadboard here, we can test the four output voltages and the frequency that is generating the circuit. Then we manufacture in a printed circuit board the oscillator we did for Reed Curtis format and the resisters that we use for the voltages that the oscillator generate. And finally we have here the final assembly for the circuit indicators.

The first step to test the electrical stimulator device is to verify the output voltage of the power supply to do this, we adjust the power supply in parallel and measure the output voltage of 12 and 12 volts between the ground and the positive and negative terminals. Once the output voltage is verified we can proceed to connect each output of the power supply in the current input of the electric stimulator device. The white cable is the ground.

The black cable is the negative voltage. And red cable is the positive voltage. To test the output signal that is generating the electrical stimulator device, we locate a culture well plate in the middle of the electrodes.

Thereafter, we connect the output voltages generated by the electrical stimulator device to each parallel plate. Given that we are working in alternated current there is no strict order of connecting the output voltage of the oscillator to the terminals of the parallel plate. To verify the output signal we use an Oscilloscope, which is connected directly to each electrode.

When the C nine is captured by the Oscilloscope we modified the amplitude and period of the signal to observe the wave completely. In this step, it is possible to verify the four voltages generated by the electric stimulator device. 50 volts, 100 volts, 150 volts and 200 volts at 60 kilo Hertz, sine wind forum.

Similar to the electric fields, a computational simulation was implemented to verify the magnetic field distribution. An axisymmetric configuration was used to simulate the coil which is composed by the cooper wire and air. Here, different material properties were considered and the applied frequency was set at 60 Hertz.

After all parameters were introduced, the model was calculated to observe the magnetic field distribution. Finally, a diagram was performed to observe how the magnetic field is homogeneously distributed in the center of the coil. The solenoid field equation, derived from Ampere's Law was used to calculate the magnetic field where is the magnetic permeability of the vacuum N'is the number of turns of the Cooper wire.

I'is the current and h'which shall be graded at its diameter, is the length of the coil. The values of these parameters were chosen to estimate a magnetic field of two millimeters. The circuit to generate the magnetic fields was computationally simulated.

Here, the transformer is connected directly to the outlet. A variable resistor was used to body the current and generate the magnetic fields of 1 millimeters long. A fuse was connected to protect the circuit.

Once calculations were performed, the polymethyl methacrylate support and the coil were built After the simulation was performed we manufactured an special device to ensure that the Petri dishes are going to be located in the middle of the stimulation device. After that, we manufacture a coin with 450 turns wire cooper in a PVC tube that is going to be located in the middle of the culture to ensure an homogenous magnetic field in the middle of the coil. After that, we manufacture a transformer with an output of six Volts and one Ampere to energize the circuit.

To test the magnetic simulator device, we measured the current that the coil is generating. This measure is performed by connecting the multimeter in series with the coil. Once we verified that the current is about one Ampere the transformer is connected to the coil to close the circuit.

Thereafter, the oscilloscope is connected to the outputs of the coil in order to verify the sine signal at 60 Hertz that is generated by the magnetic stimulation. When cell cultures are being electrically stimulated it is relevant to keep the steril conditions when culture media changes are performed to the biological samples, for this reason it's necessary to introduce the electors into the cabin. Once the culture media changed the culture well plate is located above the electrode.

To return the cell cultures into the incubator the lower electrode is located over a stable surface to place the upper electrode on the top of the cell culture. Then, the output cables of the electrically simulator device are connected to the terminus of each electrode. Finally, the electors are carefully located into the incubator to start the electrical stimulation.

Similar sterile conditions are considered when cultural media is changed to the biological samples that are being magnetically stimulate. Here, Petri dishes of 35 millimeters are used to culture either cells, explants, or scaffolds. Once culture media is changed, cell cultures need to be located into the polymethylmethacrylate support.

Here each Petri dish is located one above the other. Thereafter, the coil is carefully located over the support to cover the cell cultures. Finally, the magnetic stimulator device is located into the incubator to start the magnetic stimulation.

As you can see, the electric stimulator device has been tested to stimulate chondrocytes and osteoblasts. Here, we have evaluated the proliferation and molecular synthesis. The electric stimulator has been also tested to stimulate chondroepiphysitis, explants to assess the morphological changes in the growth plate.

Additionally, culture, mesenchymal STEM cells into uronic acid and gelatine hydrogels have been electrically stimulated to evaluate the condrogenic differentiation potential. On the other hand, their magnetic stimulator device has been tested to stimulate chondrocytes to evaluate both proliferation and molecular synthesis. The devices that we developed, in this study avoid compatibility issues counts by the electrodes when they are in direct contact with the biological material.

Moreover, these kind of devices represent an advantage because they prevent the changes in the pH and the reduction of the molecular culture gel levels. Voltage and frequencies are important variables to consider at the moment of stimulating biological tissues. On the one hand, it has been evidence that the cell dynamics such as migration, proliferation, gene expression among others, depend on the aptitude of the applied voltage.

On the other hand, it has been proved that low and high frequencies have an effect over the cells especially in the opening and closing of the cell membrane channels, which trigger different signal pathways at extra and intracellular levels. Overall, this similar device can be extrapolated to clinical environments to improve regenerative therapies such as the cellular alternative implantation. This type of treatment combines in vitro and in vivo techniques for tissue regeneration.

Here, the electric and magnetic stimulators could play a key role in the stimulation of biological materials by improving the cellular and molecular features of cells, tissues and scaffolds before being implanted in the patient.