The overall goal of the following experiment is to examine the effect of vibrational stimulation on mesenchymal stem cells using a bioreactor designed to emulate phonation. This is achieved by creating poly epsilon Capralactone scaffolds to provide a three dimensional substrate for cell cultures similar to the vocal ligament. As a second step, the scaffolds are incorporated into silicone membranes, which facilitate the transduction of the oscillation to the cell constructs.
Next laser doppler vibratory is utilized to determine the vibrational characteristics of each membrane scaffold combination. Ultimately, EISA and quantitative R-T-P-C-R analyses can be performed to assess the cellular responses at the protein and translational levels. This method can answer key questions in the field of laryngology, such as how the vocal fold matures and the consequences of vocal misuse.
The implications of this technique extend toward the treatment of vocal fold disorders because of the level of cellular control now possible with this method to fabricate the scaffold, first dissolve PCL pellets in chloroform at a concentration of 15 weight percent. Then load the resulting solution into a 10 milliliter syringe capped with a 21 gauge blunt ended needle. Lock the syringe onto a programmable syringe pump and set the flow rate at one milliliter per hour.
Then place an aluminum foil covered collector across from the needle horizontally with a needle tip to collector distance of about 18 centimeters. Next, clamp a positive alligator clip to the middle of the needle and ground the clip to the collector. Set the voltage on the high voltage power supply to 15 kilovolts, taking care to keep away from the needle.
Then turn on the syringe pump and power supply. Use a dry paper towel to quickly remove the residual polymer solution surrounding the tip of the needle before the stable fiber jet and tailor cone forms allow the fibers to accumulate on the aluminum collector for about seven hours to a thickness of 250 to 300 micrometers, and then store the result in scaffolds in a vacuum desiccate for one to two days to remove any residual solvent. The PCL scaffolds will contain micron sized interstitial pores and randomly entangled fibers with an average diameter of about 4.7 micrometers.
At a higher magnification, nanoscale grooves and pores will be visible on the individual fibers to assemble the bioreactor. Begin by using a 12 millimeter diameter biopsy punch to cut the outer diameter of an eight millimeter circular disc out of a recently spun PCL mat. Then use a second eight millimeter biopsy punch to score where the arms are to be cut.
After scoring, use a scalpel blade to cut the edges of the arms outward and insert the scaffold into the groove of the silicone membrane via the extended arms. Flatten the inserted scaffold by gently pressing the surface using flathead tweezers, and then attach an eight millimeter by two millimeter piece of thin aluminum foil to the PCL scaffold. To aid in the laser reflection, secure the assembled silicone membrane PCL scaffold in the vibration chamber.
Then sandwich the silicone membrane between paired acrylic blocks. Secure the assembly with four corner screws using a micro torque screwdriver set to a constant force of 35 cent a Newton meters, creating a watertight 24 millimeter wide and 18 millimeter deep vibration chamber. Next mounted three foot extended range mini wooer underneath the vibration chamber through another set of corner screws on the bottom acrylic block, completing the assembly of an individual vibration module.
After replicating seven additional vibration modules, affix four of the modules to one of two stationary aluminum bars by placing the speaker bases in evenly spaced circular holes. Cut into the bars, stabilize each speaker by inserting a screw through the side of the aluminum bar into each circular hole. Then connect the individual speakers to the selector by attaching wires to the positive and negative inputs on the speaker body and to the corresponding outputs on the selector.
The speaker selector allows the signal from the function generator after passing through a power amplifier to reach all eight speakers at once. Then place the two chamber arrays, the speaker selector and the associated electronics into an anti humidity enclosure, housing the entire assembly in a commercial cell culture incubator. Feed the main cables through a piece of medical grade PVC tubing and connect the power amplifier and speaker selector through the filter assembly at the back of the incubator.
To characterize the bioreactor first, add 1.5 milliliters of water into the vibration chamber to hydrate the PCL scaffold before the vibration. Then using the function generator, introduce vibration signals to the sandwiched acrylic chamber. Use a volt meter accurately.
Measure the voltage at each speaker input, and then assemble the single point laser doppler vibratory. Secure the fiber optic laser sensor. Head to a pan tilt head tripod and angle the sensor head so that it is pointing perpendicular to the tabletop.
Then connect the sensor head to the data acquisition module via a coaxial cable and the module to a laptop via the USB port. Focus the laser beam perpendicularly at various predetermined points on the silicone membrane, and then using the data acquisition software, click acquisition settings from the options menu. Next, change the measurement mode to FFT and then click the continuous measurement tool in the main toolbar.
Click the peak that forms at the chosen frequency to record the displacement and plot the normal mid membrane displacement as a function of the relative position across the substrate. To set up a vibratory cell culture first subculture human bone marrow derived mesenchymal stem cells in T one 50. Tissue culture flasks at an initial seeding density of four to five times 10 to the third cells per centimeter squared in maintenance media for seven to eight days on the day before the experiment immersive A PCL scaffold in 70%ethanol overnight.
The next morning detach the cells with Accutane and then spin down the cells and resuspend the pellet in fresh maintenance media at a concentration of 4.5 times 10 to the six cells per milliliter. After the ethanol is evaporated, expose both sides of the scaffold to germicidal UV light for five to eight minutes. Then soak the PCL scaffold in a 20 microgram per milliliter fibronectin solution at 37 degrees Celsius after one hour.
The bioreactor as just demonstrated, and then evenly distribute 40 microliters of the cell suspension onto the secured scaffold. Allow the cells to attach for one to 1.5 hours and then add an additional 1.5 milliliters of fresh media to the vibration chamber. After culturing the mesenchymal stem cell laden PCL scaffold, statically for three days, refresh the media and then impose the selected vibration regimes onto the cellular constructs.
The vibration frequencies are chosen to reflect the fundamental human speaking frequencies, and there is a linear relationship between the normal displacement and the peak tope voltage in the range of zero to 0.125 volts for all the frequencies tested at a given peak tope voltage, normal displacement decreases as frequency increases from 100 to 300 hertz. Cellular responses to vibratory stimulations examined at the mRNA levels in terms of the expression of essential vocal fold. Extracellular matrix proteins such as elastin hyaluronan synthase one, collagen type three alpha one, and matrix metalloproteinase one, demonstrate that the cyclic one hour on one hour off vibration leads to a 2.3 fold increase in elastin expression relative to the static controls.
At day seven, the vibratory stimulations also increase collagen expression moderately. It is noteworthy that the expression of major extracellular matrix remodeling enzymes, hyaluronan synthase one and matrix metalloproteinase one, is significantly augmented by the vibration signals from day three to day seven. Dynamically cultured cells produce 4.2 plus or minus 0.1 micrograms per milligram per dry scaffold weight soluble elastin as detected by Elisa after seven days of vibrations.
Whereas the static controls only accumulate 2.7 plus or minus 0.2 micrograms per milligram elastin. On average seven day, one hour on one hour off vibrations result in 2.2 and 4.7 fold increases relative to the corresponding static controls in hyaluronic acid and MMP one secretion respectively. Although our system is designed to study VOCO form physiology, it can be applied to many other systems as well.
For example, ear and human dermal tissues. Thanks for watching. Good luck with your experiments.
