Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study

The overall goal of the following experiment is to observe how changes in material properties of different layers can affect the overall mechanical properties of the graft. This is achieved by preparing natural and synthetic polymer solutions to provide optimal electro spinning parameters. As a second step, the solution should be combined in specific ratios, which will provide the desired change in material properties.

Next electro spin the solutions onto a cylindrical manl in order to create a multilayered structure with varying mechanical properties. Results are obtained that show changes in mechanical properties based on compliance, birth strength, suture retention, uni axial tensile testing, and mathematical analysis. Hi, my name is Michael McClure.

Today I'll be talking to you about tri layer to electro sponge VA of the grafts. One of the main advantages of our technique over other techniques such as singular vascular grafts is our ability to fine tune material properties within individual layers, thereby affecting our overall ME material properties Prior to electro spinning extract collagen from six month old bovine corium using an acetic acid based process. This extracted collagen is then purified through a subsequent series of dissolutions, precipitations, and dialysis collect at least 500 milligrams of lyophilized collagen for the electro spinning procedure.

Next, using a 20 milliliter scintillation vial, prepare a 100 milligram per milliliter solution of polycaprolactone by measuring 150 milligrams of monomer pellets and dissolving in 50 milliliters of 1 1 1 3 3 3 hexa fluoro, two propanol using the same procedure, prepare seven milliliters of 200 milligrams per milliliter elastin, and seven milligrams per milliliter collagen. Then use five scintillation vials and combine five total milliliters of PCL elastin collagen solutions in each vial at the following volumetric ratios, 98 to two to zero PCL elastin collagen representing the arterial intima. 45 to 45 to 10 55 to 35 to 10 and 65 to 25 to 10 PCL elastin collagen representing the arterial media and 70 to zero to 30 PCL elastin collagen for the arterial adventis.

Now using a three milliliter 18 gauge blunt tip needle load three milliliters of solution onto a syringe pump. For the individual material properties of each PCL Elastin collagen solution. Set the dispensation rate to four milliliters per hour for a total of three milliliters.

These solutions will be electro spun onto a rectangular mandrill to design multi-layered grafts with PCL elastin collagen polymer solutions. The electro spinning rate will vary depending on transition and layer electro spin, the polymer solutions for intima and four milliliters per hour and a volume of 0.5 milliliters, followed by a transition combining both intimal and medial syringes using 0.2 milliliters and electro spinning rate of two milliliters per hour each. Next, shut off the intimal syringe and electro spin the medial layer for 0.6 milliliters at four milliliters per hour.

Follow this by a transition between the media and adventis syringes of 0.2 milliliters of polymer solution at two milliliters per hour each. Finally, shut off the media syringe and continue electro spinning the adventis for 0.4 milliliters at four milliliters per hour. After electro spinning, use 50 millimolar EDC on each sample and allow 18 hours for cross-linking before further testing.

To test electros spun materials for uni axial tensile parameters First hydrate the electros spun sheet in 15 milliliters of one times PBS for 24 hours in a Petri dish. Then using a dog bone punch with a gauge length of 7.5 millimeters and a 2.5 millimeter width at its narrowest point, punch six samples from the electro spun mat. This shape will ensure material failure at its narrowest point.

Next, using an MTS Bionics 200 testing system, test the electrodes BUN samples with a 100 N load cell and a 10 millimeter per minute extension rate. Use the test work software to calculate peak stress, modulus and strain at break for each individual material product. To test suture retention of multilayered grafts, collect six different two millimeter in a diameter electro sponge, tubular specimens, three millimeters in length, and soak the specimens in a Petri dish with one times PBS for 24 hours in an incubator at 37 degrees Celsius.

Then use a 50 N load cell and an extension rate of 150 millimeters per minute. To test suture retention with the MTX bionic 200 system, place a five zero commercial PDs. Two violet monofilament.

Suture two millimeters from the end of the sample using a pair of calipers to measure and extend until the suture pulls through the graft record peak load in gram's force using test works. Version four, to test birth strength on electros spun grafts, collect another six different two millimeter in a diameter electros spun tubular specimens two to three centimeters in length and hydrate in PBS for 24 hours at 37 degrees Celsius. After hydration, fit the tube over a 1.5 millimeter diameter nipple attached to the device and secure with a two zero silk suture by tying a surgeon's knot around the graft and nipple End, then introduce air into the burst system and increase the pressure at a rate of five millimeters of mercury per second until the tubes burst.

Collect data on burst strength as pressure in millimeters of mercury at which the tubes rupture. Finally, evaluate dynamic compliance by obtaining six two millimeter in a diameter tubular grafts taken from six different electros spun grafts, cut to a length of three centimeters. Hydrate samples in PBS for 24 hours.

At 37 degrees Celsius. Fit the grafts over a custom made 1.5 millimeter diameter nipple securing with a two zero silk suture by tying a surgeon's knot. Place the grafts on an intelligent tissue engineering via mechanical stimulation bioreactor developed by tissue growth Technologies.

Fill the bioreactor with 500 milliliters of PBS at 37 degrees Celsius. Set the bioreactor to provide a one hertz cyclic pressure change to the inside of the graft and test three different pressure levels of 90 over 50, 120 over 80 and 150, over 110 millimeters of mercury for systolic diastolic pressures. When the electro spitting protocols are carried out correctly, the end product should be a soft seamless tube as seen here with no initial signs of deamination between the layers.

A delaminated tube is shown here for comparison. This image provides information on the UNI axial tensile test values for peak stress tangential and strain to break for individual intimal, medial and adventitial layers containing specific PCL elastin collagen blends. The trends indicate increasing peak stress and tangential modulus with increases in PCL content while strain to break decreases suture retention results are shown here with E-P-T-F-E displaying the highest suture retention and 65 to 25 to 10 being closest to the ous vein standard shown as a dash line non-viable pig femoral artery and E-P-T-F-E were tested as a comparison.

Burst pressure as shown in this graph, shows that 55 to 35 to 10 and 65 to 25 to 10 materials have the highest burst pressure. And this is similar to the 1, 600 millimeters of mercury that the American National Standards Institute requires for all vascular grafts intended for market use. Finally, compliance tests determined from internal radar at three different mean arterial pressures indicate how increased PCL content in the medial layer alone can cause a gradual decrease in compliance for all three types of physiological pressures, The most critical portion of this study is the legislating process.

When using a three to one input output, nozzle arkin in charge loss may occur. If this does, the bulge is associated with a polymer that is currently being electric spun can decrease as well. This can cause welded wet fibers and delamination between the three layers.

Therefore, a consistent electric potential is essential when electric spinning a tri later vascular graft.