The overall goal of this experiment is to synthesize and characterize keratin-base nanofiber for biomedical application. This method can help answer key questions in the biomedical engineering field, such as wound healing, drug delivery, and engineered tissue, such as liver, bone, and muscle. The main advantage of this technique is that it is cheap, efficient, and can be upgraded to large-scale production.
The implication of this technique extend to a treatment of wounds and delivery of drug for tissue regeneration. Though this method can provide insight into the advantage of using electrospun nanofibers, it can also be applied to other systems, such as tissue regeneration. Generally, individuals new to this method will struggle because it is difficult to make a polymer substance that can generate nanofibers.
Procure approximately 20 grams of human hair clippings that have not been chemically treated or altered. Hair can be any length. Wash the hair thoroughly by hand with warm water and soap.
Use deionized water for the final rinse. Put the hair into two 600 milliliter beakers and place in an 80 degree Celsius oven for one hour. Once dry, divide dry hair evenly between the 600 milliliter beakers, placing 10 grams of hair into each beaker.
Do not allow the hair to fill more than 500 milliliters. Next, pour 500 milliliters of peracetic acid solution, or PAS, into each beaker, making sure to cover all of the hair. Cover the beakers with Parafilm and store for 12 hours.
Separate the hair from the PAS using a 500 micron sieve, collecting the waste PAS in a separate container. Rinse the hair thoroughly with deionized water to remove any leftover PAS. After placing the rinsed hair in a 500 milliliter flask, pour 400 milliliters of 100 millimolar Tris Base solution, or TBS, into the flask, making sure that the hair is covered.
Then, place the flask in a shaking bath set to 38 degrees Celsius, 65 RPM, for one hour. After one hour, remove the flask from the shaking bath. Pour out the liquid, which is approximately 400 milliliters of keratin-extraction solution, or KES, into a 1000 milliliter beaker.
Pour 400 milliliters of deionized water into the flask containing the hair, ensuring that the hair is covered before placing the flask back in the shaking bath for one hour. Remove the flask from the shaking bath and pour the remaining 400 milliliters of the KES into the 1000 milliliter beaker with the previously collected KES. The hair is no longer needed and can be discarded in the nearest trash receptacle.
After neutralizing the collected KES as described in the text protocol, pour the solution into the 500 milliliter round bottom flask until about a quarter full. Run the distiller according to the manufacturer's protocol for 1.25 hours, ensuring that the flask is connected tightly. Repeat this process until all of the KES has been distilled.
Pour the distilled KES solution equally into 12 separate 14 milliliter conical tubes. Centrifuge the tubes at 1, 050 times G for 10 minutes. Pour the centrifuged KES into a clean beaker, making sure to pour away from the collected debris.
Repeat until all of the KES has been centrifuged. Next, cut dialysis tubing cellulose membrane to 24 inches and clip one end of the tubing to hold it shut. Open the non-clipped end of the dialysis tubing cellulose membrane and slowly pour 60 milliliters of centrifuged KES solution into the tubing.
Use another clip to close this end of the tubing. Put the dialysis tube in a 2, 000 milliliter cylinder of deionized water and allow it to sit for 24 hours, changing the deionized water in the cylinder every three to four hours. Then, empty the dialyzed KES solution into capped jars, being sure to leave space at the top.
Place the capped jars into a minus 20 degree Celsius freezer for 24 hours. Following the 24-hour freezing period, take the caps off of the jars and place them into freeze dryer ampules. Place the seals on the ampules and turn the knob to create a vacuum pressure of 0.133 millibar.
Lyophilize the samples for approximately 48 hours until all of the moisture is gone. Prepare a PCL keratin solution by adding a 10 weight keratin solution into a 10 weight PCL solution drop wise in order to create 10 milliliter solutions with PCL to Keratin ratios of 90 to 10, 80 to 20, 70 to 30, and 60 to 40. Place approximately eight milliliters of the vortexed PCL/keratin solution into a 10 milliliter disposable syringe, fitted with a 0.5 millimeter diameter plastic tube.
Place the syringe in a syringe pump where the flow rate is set to be 2.5 milliliters per hour. Apply voltage to the tip of the needle connected to the tube. Apply a 19 to 22 kilovolt voltage to the tube and rotate the collector drum at around 200 RPM.
Cut the fibers into 16 square millimeter samples. Use a biocompatible silicone-based glue to fix each sample to cover slips with a 12 millimeter diameter. Glue one end of the fiber sample to the back of the cover slip and wrap the sample around the cover slip.
Then, glue the free end of the sample to the back of the cover slip as well, leaving the top of the slip covered with only the fiber sample. Place the fiber samples in a 24 well plate. Sterilize the samples by immersing them in 80%ethanol for one hour.
Rinse the ethanol from the samples using deionized water. Then, pipette 2 milliliters of basal medium onto the samples, making sure to cover the entire sample for five minutes. Use fibroblast 3T3 cells to seed the fiber samples.
Suspend the cells in DMEM, supplemented with antiobiotics and 10%fetal bovine serum so that there are approximately 62, 000 cells per square centimeter per one milliliter of DMEM. Drop one milliliter of the 3T3 cell containing DMEM solution into each well plate, ensuring that each fiber sample is covered with approximately 62, 000 cells per square centimeter. Incubate the well plates for 24 hours at 37 degrees Celsius and 5%carbon dioxide.
After cutting the dried PCL/keratin nanofiber membranes, sterilize the 900 square millimeter samples with 80%alcohol for a 10 minute incubation period. Then, wash the membranes thoroughly with deionized water. Next, incubate the samples in 15 milliliters of PBS, pH 7.5, at 37 degrees Celsius, replacing the buffer every three days.
Take the membranes out of solution at specified intervals of one week and seven weeks. Rinse with deionized water. Place the membrane samples into freeze dryer ampules.
Place the seals on the ampules and create a vacuum to lyopholize the samples for 24 hours until completely dry. Next, attach the dried sample to a scanning electron microscope, or SEM, stage using copper tape. Place the stage inside the sputter coater and coat with golds at 15 milliamps for one minute and 30 seconds.
Load the sample into the chamber of the SEM. Observe the fiber samples at an accelerating voltage of 1.5 kilovolts and five microamps of current. Representative images of PCL/keratin-based nanofibers successfully electrospun from solutions with PCL/kertain ratios of 70 to 30, 80 to 20, and 90 to 10 are shown here.
The electrospun fibers were found to have Young moduli close to that of the native tissue. The Young's moduli decreased with increased keratin ratio. The figure displays a graphical trend of variation of Young's modulus versus PCL/keratin ratio.
The Fourier transform infrared spectroscopy spectra displayed here confirms the bonding of PCL to keratin. The major peak, measured at 1, 722 inverse centimeters, agrees with the standard basic measurements of the PCL absorption band and is visible in all spectra. Cell adhesion and proliferation on the PCL keratin fibers confirms that the fibers are not toxic and provide support for cell growth.
The filopodia growth along the nanofibers indicates favorable interaction between the fibroblasts and the PCL/keratin fibers. Once mastered, this nanofiber synthesis technique can be done in 24 hours if it is performed properly. While attempting the electrospinning procedure, it's important to remember the viscosity of the polymer solution, flow rate of the solution, voltage applied, speed of the rotating drum, and steps for fibroblast cell culture.
After watching this video, you should have a good understanding of how to synthesize in PCL/kertain-based nanofibers and its importance in biomedical engineering field. Following this procedure, other methods like cell viability and in vivo degradation testing can be performed in order to answer additional questions about the toxicity response to tissue, tissue growth, and the degradation of the fibers. After its development, this technique paved the way for researchers in the field of regenerative engineering to explore fields just as skin regeneration, wound healing, and drug delivery.
Don't forget that working with organic solvents, such as TFE, and high voltages can be extremely hazardous and precautions such as wearing safety glasses, lab coats, and gloves, and proper disposal of solvents should always be taken while performing this procedure.
