Triple-layered structures consisting of orientated fibers can be found all over the human body. Applying this method, one cannot only create heart valve leaflets mimicking natural conditions, but also a variety of other tissue. This is the first time 3D printed collectors out of conductive material have been used in electrospinning, a fact that makes this process highly flexible and cost effective.
To begin, start 3D printing by uploading the STL file specimen mount A and specimen mount B into the slicing software. Rotate the models so the triangular surfaces are placed on the build plate. Mark all parts, right-click, and select multiply selected models.
Enter one in the prompt number of copies and click OK.Set slice thickness to 0.1 millimeters, wall thickness to one millimeter, infill density to 40%and uncheck the generate support box. Click the slice button and then select save to removable to save the printing file to a USB drive. Keep the settings and replace the STL files with collector flange and leaflet template in the slicing software.
Use the copy tool to create one copy of the flange and eight copies of the template before starting the print. After completion of the print, remove the models from the build plate. Remove individual filament fibers at the bottom of the leaflet negative carefully with a wire cutter if these are present in the leaflet models.
To prepare a spinning solution, place a scale under the exhaust hood. Position a 200 milliliter screw cap glass bottle on it and tare the scale. Pour 50 milliliters of dimethylformamide and 50 milliliters of tetrahydrofuran into the glass bottle.
Note the weight of the solvents. Place a magnetic bar inside the bottle. Place the bottle on a magnetic stir and switch it on.
Transfer the corresponding amount of polyurethane slowly into the glass bottle containing the solvent mixture while stirring at room temperature to obtain a homogeneous solution. Afterwards, close the lid. Assemble the 3D printed parts together with the metal bars to create the collector and make sure that all templates are oriented correctly.
Place the assembled collector in the electrospinning setup and tightly secure the flanges to the motor axis. Using a crocodile clip, connect the cable connected to the cathode to the 14 gauge needle and check the connection between clip and needle. Connect the collector to the anode using a crocodile clip and the second high voltage cable.
Use a slip ring or a stripped cable to create contact at the collector's flange. Prepare a Luer lock syringe by filling it with 20 milliliters of the spinning solution. Connect the syringe to the solvent-resistant tube and manually push the solution into the tubing system until a droplet is visible at the tip of the needle.
Place the syringe in the syringe pump. After turning on the pump, set the diameter at 19.129 millimeters, volume at five milliliters and speed at three milliliters per hour. To test run the motor, connect to the motor control by clicking the connect button.
After connecting, select the profile velocity operation mode and click on the operation tab located in the upper left corner of the screen. Select the profile velocity tab below the quick stop button framed by a red line. Then set the target velocity of 200 RPM, profile acceleration of 100, profile deceleration of 200 and quick stop of 5, 000.
Start the test run and check the collector for any unbalance. Stop the motor by clicking the switch on enabled button and change target velocity to 2, 000 RPM. To manufacture the layer in the motor control software, click the enable operation button to switch on the motor.
Switch on the high voltage power supply and adjust the voltage for both anode and cathode with the minus pole as 18 kilovolts and the plus pole as 1.5 kilovolts. Start the syringe pump at a flow rate of three milliliters per hour. Observe the needle tip for the formation of a Taylor cone and depending on the shape of the cone at the needle tip, adjust the voltage at the cathode in increments of 100 volts until a stable Taylor cone is established.
Stop the spinning process by switching off the power supply unit, syringe pump, and motor. Then change the target velocity to 10 RPM in the motor control software and repeat the layer manufacturing process as described previously for another 20 minutes. After the second layer has been added, carefully open the screws connecting the collector flanges to the motor axis and remove the leaflet collector from the electrospinning device.
Using a scalpel, cut the electrospun fibers along the outer contour of each leaflet template. Remove the flange on one side of the collector. Then pull out the 3D printed inserts and separate the leaflet templates from the non-conductive triangular holders.
Rotate all leaflet templates by 90 degrees and reassemble the collector. Insert the collector into the electrospinning setup and tightly secure it. Again, change the target velocity back to 2, 000 RPM in the motor control software and start the layer manufacturing process as described previously for 20 minutes to add the third layer of fibers.
After removing the collector from the electrospinning device, dry the samples in a heating cabinet at 40 degrees Celsius. After the samples are completely dried, use a scalpel to carefully cut along the edges of the leaflet template to remove surplus fibers. Afterward, carefully peel the leaflet scaffold of the template and place it on a tray for further use.
A triple-layered leaflet scaffold mimics the collagen configuration of the native human heart valve and each layer consists of fibers with a diameter of approximately 4.1 micrometers. Scanning electron microscopy imaging revealed aligned fibers with smooth surface and strict orientation in the circumferential direction, while unaligned fibers showed disordered orientation and many prominent intersections between fibers. Fluorescence imaging revealed that the bottom layer consists of aligned fibers in horizontal orientation with the very little intersection between the fibers.
The middle layer shows unaligned fibers with no primary fiber orientation, while the top layer shows aligned fibers in a perpendicular orientation. Thickness measurement shows a linear increase in thickness of approximately 2.65 micrometers per minute. After 60 minutes, an approximately 2.52 micrometer per minute increase in thickness was observed.
Tensile tests for aligned fiber scaffolds have a strength of approximately 12 and 3 Newton per millimeter square along the circumferential and perpendicular orientation. However, unaligned fiber scaffolds show no difference in the tensile strength for different orientations. The aligned fiber scaffolds revealed extensibility of approximately 187 and 107%in the circumferential and perpendicular direction, while the unaligned fibers revealed uniform extensibility in both directions.
Stress strain curves showed that the unaligned fiber mats exhibit linear elastic behavior, while aligned fibers revealed nonlinearity in the axial direction. The created leaflets can be used for biological and biomechanical assessment. When three of them are assembled to create a functional aortic valve, a wide range of in vitro experiments can be performed.
The protocol will enable fellow researchers not only to manufacture multilayered fiber scaffolds, but also to orientate fibers. Therefore, they will be able to mimic many different types of tissue.
