This protocol reported the transformation of traditional electrospun nanofibremembranes from 2D to 3D, via depressurization of subcritical CO2 fluid which was not realized previously. This method eliminates many issues associated with previous approaches, including the use of aqueous solutions and chemical reactions, multiple step processes, loss of activity of encapsulated biological molecules, and limitations of hydrophobic polymers. Demonstrating this procedure is Shixuan Chen, a postdoc from my lab.
In a 20 mililitre glass tube, dissolve two grams of PCL in a solvent mixture of dichloromethane and DMF, with a four to one ratio at a concentration of 10 percent. Place the glass tube into a lab rotator until the solution becomes clear. The solution may mix over night.
To set up the Electrospinning Apparatus, first, add the PCL solution to a 20 milliliter syringe with a 21 gauge blunt needle attached. Ensure there is no air in the syringe, and dissociated tubing. Place a rotating steel drum with the ground collector 12 centimeters from the needle tip.
Using alligator clips, connect the direct-current high-voltage power supply to the needle, and ensure that the collector is grounded. For the 20 milliliters of PCL solution, set the parameter of the syringe pump using a diameter of 20.27 millimeters, and a flow rate of 0.5 milliliters per hour. Check if the droplets are forming at the tip of the needle.
Apply an electric potential of 20 kilovolts between the spinneret and a ground collector located 20 centimeters away from the spinneret. Collect the aligned nanofiber mats in a drum, rotating at 2, 000 RPM. Collect the PCL nanofiber mats once they reach a thickness of approximately one millimeter.
Immerse the PCL nanofiber mats in liquid nitrogen for five minutes. Keep the PCL nanofiber mats in liquid nitrogen, and punch PCL nanofiber mats with a 0.5 millimeter diameter punch. Place the PCL nanofiber mats into liquid nitrogen for five minutes.
Cut the mats into one centimeter by one centimeter squares using sharp surgical scissors while submerged in liquid nitrogen to avoid deformation of the edges. Place the cut mat in a 30 milliliter centrifuge tube with approximately one gram of dry ice. Tightly cap the lid, and allow for the dry ice to change into liquid carbon dioxide.
Once the liquid has formed in the tube, quickly release the pressure by opening the cap. Remove and observe the puffed scaffold from the tube. Place the scaffold in a new centrifuge tube with dry ice, and repeat util the desire thickness is achieved.
Sterilize the expanded nanofiber scaffolds in ethylene oxide prior to incubation with cells. The effectiveness of expanding traditional 2D electrospun nanofiber mats into 3D scaffolds via depressurization of subcritical CO2 fluid is shown on the left after the second treatment. The thickness of the scaffold increased from one millimeter when untreated to 2.5 millimeters with one CO2 treatment, to 19.2 millimeters with two CO2 treatments.
The porosity of the scaffolds increased from 79.5 percent for the untreated mats, to 92.1 percent after the first treatment, to 99.0 percent after the second treatment. This is significant because the degree of cell penetration into a scaffold, and thus its efficacy to induce regeneration, is largely dependent on the porosity. SEM images reveal that the densely-packed fibular structure of untreated 2D mats were transformed into ordered, layered structures with aligned nano fibers after expansion with CO2.
In Vevo studies were carried out by subcutaneous implantation of CO2-expanded nano fiber scaffolds with square-arrayed holes to rats. This allows for cellular migration and proliferation within the holes, as well as further infiltration within the nano fiber layers that were created during expansion. From week one to four post-implantation, the expanded scaffolds showed a significant increase in the number of blood vessels formed, and multinucleated giant cells when compared to a traditional nano fiber mat.
Following this procedure, different molecules including growth factors, amino-modulating compounds, hemostatic agents, and anti-microtubular agents can be incorporated into the nano fiber mats and expanded in subcritical CO2 fluid. Such functionalized expanded nano fiber scaffolds could be used to explore new questions in other scientific fields, such as hemostasis, prevention and treatment of infection, immunology, and tissue regeneration and repair. Organic solvents are toxic, and should be handled in a chemical hood.
Furthermore, a container that can endure the high pressure of subcritical CO2 fluid should be used for the expansion.
