The overall goal of this procedure is to create a multifaceted environment to support nerve growth and repair. This is accomplished by first encapsulating growth factors within degradable microspheres. The second step is to prepare the support material for the bulk of the environment.
Next, the microspheres and support material are combined and electro spun into an aligned fibrous scaffold. The final step is to test the scaffold with chick dorsal root ganglia neurons. Ultimately, immunofluorescence microscopy is used to show that neurite growth and direction is accelerated compared to controls.
Though this system is designed for neural tissue with slight changes to the proteins and materials used, it could also be applied to various tissue types, including hearts, lungs, and bone. Prior to starting this procedure, prepare the necessary reagents, 2%and 0.5%weight per volume solutions of polyvinyl alcohol or PVA in deionized water, a solution of 2%volume per volume, isopropyl alcohol and deionized water, and an aqueous solution of the desired hydrophilic protein.Place. 40 milliliters of the 0.5%PVA solution into a 50 milliliter centrifuge tube and set aside in a small vial dissolved 300 milligrams of 65 to 35 polylactic co glycolic acid or PLGA and three milliliters of di chloro methane.
A vortex mixer can be used to accelerate PLGA dissolution combined 200 microliters of protein solution and four microliters of 2%PVA solution. Pour the protein PVA mixture into the PLGA solution. The solutions will remain mostly separate.
Place the vial into a beaker of ice water using a W ator at about 10 watts, agitate the solution for five to 10 seconds until a uniform creamy white emulsion is created. Pour the emulsion into the previously prepared 50 milliliter tube containing 0.5%PVA. Mix the solution at high speed on a vortex mixer for about 20 seconds.
The solution will develop a cloudy appearance. Transfer the emulsion to a 200 milliliter beaker and place on a stir plate at 350 RPM for two minutes. Add 50 milliliters of 2%isopropyl alcohol to the beaker on the stir plate.
Allow the mixture to continue stirring for a minimum of one hour to allow the chloro methane to evaporate and the PLGA to harden. Next, transfer the microsphere solution into centrifuge tubes, centrifuge at 425 times G for three minutes. The microspheres will collect at the bottom of the tube and appear white Carefully remove the supernatant from the tube above the microspheres and store in a 500 milliliter bottle.
Rinse the microspheres with deionized water by filling each tube three quarters full and shaking it to redistribute the microspheres in the liquid. Repeat centrifugation removal of the supernatant and rinsing of the microspheres with deionized water four times following the final rinse. Remove the supernatant and place in the 500 milliliter bottle with the other samples.
Freeze the microspheres collected in the centrifuge tubes at negative 20 degrees Celsius overnight, and then lyophilize for at least 24 hours. The following electro spinning solution should be prepared beforehand in deionized water, 2%weight per volume, meth related hyaluronic acid or miha with 3%weight per volume, 900 kilodalton polyethylene oxide or PEO and 0.05%weight per volume. Photo initiator solution.
After making the desired volume of electro spinning solution, add microspheres at the desired concentration up to 400 milligrams per milliliter. Mix the solution on a vortex mixer until the microspheres are evenly distributed in the solution. Transfer the solution to a syringe and attach a six inch 18 gauge blunt tip needle.
Place the syringe in a syringe pump and set it to dispense at 1.2 milliliters per hour. Tape a layer of aluminum foil on the mandrel. This allows for easy cleanup and storage of the finished scaffold.
A rotating mandrel is used to create aligned fibers. Connect the ground wire from a high voltage power source to the collection apparatus. Connect the positive lead to the needle To ensure successful electro spinning.
It's very important to verify that all connections and settings are correct. Start the polymer pumping and when the solution is visible at the end of the syringe, turn on the voltage source and set the voltage to 24 kilovolts. Run the solution until the desired scaffold thickness is achieved.
When complete, turn off the voltage source and syringe pump. To begin this procedure, attach related cover slips to the collection area of the electro spinner with removable double-sided tape before electro spinning. Spinning onto the cover slips, eases handling and viewing after electro spinning to the desired thickness.
As demonstrated in the previous video segment, carefully remove the cover slips from the mandrel. Place the scaffold coated cover slips into a clear nitrogen chamber and ensure that all oxygen is purged. Place the chamber and scaffold under a 10 milliwatt centimeter squared 365 nanometer light for 15 minutes.
After cross-linking place, cover slips into an appropriately sized well plate. Ensure that the scaffold side is facing up. Place 100 to 200 microliters of media on each scaffold in the well plate.Carefully.
Place one dorsal root ganglia or DRG on each scaffold in the media droplet. For a thick scaffold, more media may be needed. The DRG needs to be fully submerged and not floating.
Incubate the scaffold and DRG at 37 degrees Celsius for four hours to allow the cell to adhere to the scaffold. Next, fill media to the appropriate level for the well. Return the plate to the incubator and incubate for four to six days after the incubation period.
Carefully remove the media from each well and gently wash once with PBS. Fix cells for 30 minutes using 4%weight per volume. Para formaldehyde subsequently stain the cells for neuro filaments and nuclei following an established protocol.
To visualize the cells using a fluorescent microscope, place the well plate on the stage of the microscope and locate the cell mass using the filter and excitation settings for dpi. Once the cell is identified, switch the filter to zi. To visualize the extended neurites, use the stitch function on the microscope to collect and combine as many images as necessary to see the entire structure.
Repeat for dpi, fite and brightfield microspheres. 50 plus or minus 14 micrometers in diameter with over 85%protein encapsulation have been consistently produced and electros spun into scaffolds by this protocol. This fluorescent image shows representative microspheres with Rod Domine in the PLGA shell and BSA fits encapsulated within.
To evaluate encapsulation, the microspheres were filled with BSA and tested for protein release for over 60 days with a Bradford protein assay. The release begins with an initial burst and then continues as microspheres break down after electro spinning. The microspheres can be seen throughout the 3D fibrous structure.
In this SEM image of the complete scaffold. The spheres can be seen in multiple layers indicated by arrows. This high magnification image is of one microsphere with the nano fibers from the scaffold above it.
Dorsal root ganglia or DRG were used to test the viability of nerve growth factor or NGF in the microspheres within the scaffold. Here are DRG seated on a scaffold containing microspheres. Loaded with NGF is shown next to one microspheres with no protein within the longer neurite Extensions extending from the DRG on the NGF scaffold indicate that the NGF is viable and can promote growth.
After watching this video, you should have a good understanding of how to encapsulate protein into microspheres and incorporate them into fibrous scaffolds.
