This fabrication technique allows for the production of nanofiber scaffolds with a gradient in the organization of the Fibers. Gradated organization offers increased utility of nanofiber scaffolds in the biomedical field. Electro spinning offers the capability to produce nanoscale fiber scaffolds with large surface areas.
Fabrication takes place by combining a polymer with a volatile solvent, then moving that solution at a constant rate through a syringe with a high voltage applied charges the solution and elongates the polymer as it is released from the needle tip. The result is a dry nanoscale polymer fiber that can be collected in a number of ways for various applications. In this protocol, we spun a polycaprolactone polymer onto a gap collector to form a ally aligned fiber substrate.
Then we used a gradually moving mask To cover this substrate while random PCL fibers were deposited. Hello, my name is Carl Kalala. From the laboratory of Dr.Xing.
We jia in the Department of Pharmaceutical Sciences and Holland Regenerative Medicine Program at the University of Nebraska Medical Center. Today we'll be, we will be showing the protocol for the fabrication of a nanofiber scaffold with an organizational gradient. Additionally, we'll be showing the biomedical applications of these scaffolds through nano Encapsulation and stem cell seeding.
Firstly, ensure proper safety equipment is worn When dealing with potentially hazardous chemicals. A, the primary step in this procedure is to prepare a solution of polycaprolactone or PCL at an approximate concentration of 100 milligrams per milliliter. Dissolve the polycaprolactone in a mixture of di chloro methane and dimethyl for amide at a ratio of four to one volume over volume for each respective solvent with an overall concentration of 10%weight by volume for the solution mix until the solution is translucent and homogeneous with an appropriate viscosity.
Add the PCL solution into a five milliliter syringe with a 21 gauge blunt needle. Attached place a single syringe pump in a vertical electro spinning position with the collector directly below the syringe pump ground. The collector ensure that the collector is individually ground with no contact to the other lab equipment.
For efficient fiber deposition, set the syringe pump to a relatively high flow rate to prime the needle for electro spinning. Run the pump until droplets forming at the needle tip are immediately replaced. When removed, for electro spinning, set the pump to 0.5 milliliters per hour.
Turn the voltage operator to 12 kilovolts. Caution as this high voltage can be very dangerous to avoid any proximity. With the needle tip, we use a non-conductive extension to handle any potential blockages or extraneous fibers that may occur on the needle tip or at the collector.
Fibers should start to appear across the gap on the Collector. Continue electro spinning until a relatively thick fiber mat has been fabricated. Across the gap on the collector, apply glue to the edges of a small glass plate.
The plate dimensions should be slightly smaller than the gap collector with the aligned fibers, so the scaffold is effectively removed. Remove any extraneous fibers that may have accumulated on the front of the collector. Then place the collector on the glass so the fiber mat makes full contact with the plate.
Pad, the scaffold lightly to ensure the fibers are directly contacting the glue. Let the scaffold sit until the glue is dried. Once the glue is dried, carefully cut around the glued edges of the scaffold, so a section of the aligned mat remains immobilized on the glass plate.
The result should look something like this with the fiber scaffold immobilized on three sides for secondary deposition. If chemical encapsulation is desired, add Kumar and six. Add a concentration of 1%weight by weight to the PCL solution Mix until the solution is homogeneous and exhibits a fluorescent green color for gradient preparation.
Position the second syringe pump perpendicular to the first pump and collector. Attach a plastic mask to the second syringe pump and position it approximately two millimeters above the collector. Prep the needle using a high flow rate.
When fibers begin to deposit above the collector, set the horizontal syringe pump to pull at nine milliliters per hour or an approximate Moving velocity of one millimeter per minute. As the experiment progresses, more Random fibers should be visible on the aligned scaffold. Dyes such as Kumar and six greatly enhance this visual gradient representation.
As fiber deposition progresses, the nana fibers will begin to deposit outside of the desired range of the collector. Remove these fibers using a non-conductive rod so they do not interfere with the fiber scaffold electros spin until the mask has moved almost completely off the collector. Make sure to leave a small amount of the aligned fiber scaffold under the mask.
The finished scaffold should have distinct aligned and random constituent parts visualized here with the green random deposition and the white aligned fibers, A gradual change from random to uniaxial fiber alignment should be evident in the Intermediate area of the scaffold. Perform a standard cell culture with the a depose Derived stem cells. Then seen the cells on the fiber scaffolds and incubate at 37 degrees Celsius for two hours.
After two hours of incubation, apply more cell culture to the fiber scaffolds until they're sufficiently covered in the medium. Then incubate two samples at 37 degrees Celsius for Three and seven days respectively. These are scanning electron microscope Images taken at two millimeter increments.
On the scaffold. There is a clear progression from aligned to random fibers on the scaffold. The four year fast transfer patterns reaffirm this progression as the pattern at zero millimeters indicates alignment, and the pattern at six millimeters indicates a random orientation from the stem cell culture.
Two sets of fluorescent microscopic images were taken. They both indicate different cell orientations, which are mediated by the position of the seated cells. On the alliance scaffold.
70%of the cells appear within 20 degrees of the axis of fabrication compared to only 20%of the cells in the random portion of the scaffold. The formation of a chemical gradient through nano encapsulation is examined by fluorescent microscopic images. The graph of the fluorescent intensity shows a linear growth indicating a gradual change in the chemical concentration through the scaffold.
Chemicals such as bone morphogenetic protein two can be encapsulated in this fashion To promote osteoblastic differentiation for tissue engineering, our final goal is to develop a tissue construct, which will combine a unique class of scaffolds with dual gradations in structural organization and mineral content. This combination can fully recreate the native tendon to bone insertion site. Incorporating a D post derived stem cell seeding should allow for Enhanced repair of rotator cuff injuries.
Today we have introduced the protocol for the production of a nanofiber Scaffold with a gradient in the organizational structure and explained its potential biomedical applications. The most critical portion of this protocol is the random secondary deposition on the aligned scaffold to create the organizational gradient during the gradient preparation, carefully watch the needle tip and the collector remove any globs at the needle tip or extraneous fibers at the collector to ensure a uniform scaffold.
