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Melt Electrospinning Writing of Three-dimensional Poly(ε-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications
Chapters
Summary December 23rd, 2017
This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.
Transcript
This video tutorial reflects on electrospinning with polymer melts in a direct writing mode and provides necessary guidelines to fabricate scaffolds with well ordered architectures. Electrospinning refers to a polymer processing technique where a viscous polymer is extruded through an orifice while the application of an electrical field provokes the the rise of a jet. Electrostatic forces accelerate the fiber towards an oppositely charged or grounded collector.
In order to reach a required level of viscosity, polymers were traditionally liquified in solvents. These evaporate during the flight phase, which, in combination with the electrical field, provokes strong buckling of the fibers. This results in polymeric scaffolds whose morphological properties are challenging to control, characterize, and reproduce.
This experiment demonstrates electrospinning with polymer melts. Instead of dissolving, the required level of viscosity is reached by the application of heat to virgin polymers. The lack of solvents improves the macromolecular entanglement of the polymer and results in a more stable fiber flight path.
The micron scale fibers can be accurately deposited according to the movement of the collector. Electrospinning with polymer melts in a direct writing mode allows for the fabrication of well ordered scaffold architectures, down to the micron and even nano scale. We predict that the technology will play a key role in the future of tissue engineering and regenerative medicine.
The technology bridges a long-awaited gap between conventional fiber-forming processes and 3D-printing principles and opens up new avenues for designing the next generation of scaffolds. Micro electrospinning writing facilitates the direct processing of medical-grade plastics, such polycaprolactone or PCL. Because of the small diameters and highly organized structures and porosity, melt electrospun PCL scaffolds are ideal structures for this as in vitro and in vivo.
One major prerequisite for achieving an effective pyrolysis with valuable results is a highly functional soft-and hardware. Therefore, we engineered a fully integrated system with automated perimeter control. This facilitates a reliable and specifically reproducible production of scaffolds to sufficiently meet the requirements of the users.
Melt electrospin writing involves manufacturing scaffolds with different architectures. The following tutorial describes material preparation, scaffold production, fiber diameter adjustment, and optimization of the jets. Fill two grams of PCL in a three milliliter plastic syringe and push the plunger in gently.
Place the syringe in a preheated oven for eight hours at 65 degrees Celsius. Carefully squeeze the plunger until all the trapped air bubbles are removed. Connect a 23ga needle and attach it to the air pressure system.
Press it down until the needle tip emerges from the brass pot. Install the collector on the stage. Mount and clean it with 70%ethanol.
Determine speed and direction of the collector movement by programming the G-code to vary size, spatial fiber distance, and number of layers of the scaffold. For tubular scaffolds, select rotational speed, translational speed, and its repetitions to define the thickness of the scaffold. A detailed guideline to individually program G-codes for flat or tubular scaffolds can be found in the manuscript.
Open the software Mach3 and upload the generated G-code. There are five adjustable system parameters which significantly determine the quality of the resulting scaffold. These are air pressure, voltage, collection speed, temperature, and working distance and should be adjusted in the following order.
Place a 12-millimeter high 3D-printed plastic block between needle and collector to adjust the working distance. Switch on both heaters and set to 65 degrees for the top and 82 degrees for the bottom part. Initiate the air pressure and wait until the molten polymer is extruded from the needle.
Before starting the process, ensure that all grounding cables are connected firmly. Close the front door of the enclosure which connects the interlock. Increase the voltage until the extruded melt transforms into a Taylor cone and ejects a molten jet towards the collector.
Allow the polymer melt to be extruded on the still collector plate for five minutes to stabilize the jet. Electrospinning with polymer melts allows for the fabrication of different fiber diameters. This can be achieved via specifically varying the system parameters pressure, voltage, and speed, whilst maintaining distance and temperature on a constant level.
Small-diameter fibers between three and 10 micron can be printed via applying a relatively low pressure, small to medium electrical field, and high collection speeds. Based on the experience in our lab, we can print four micrometer diameter fibers with the following values:0.9 bar, eight kilovolt, and 1, 700 millimeters per minute. Medium-sized diameters between 10 and 20 micron can be printed via applying relatively low pressure level, medium to high voltage, and collection speeds in the medium range.
Based on the experience in our lab, we can print 13-micrometer diameter fibers with the following values:1.5 bar, 11 kilovolt, and 1, 200 millimeters per minute. Fibers in the range of 20 to 30 micron or more require large polymer extrusion rates alongside the application of comparably high voltages and the lowest possible collection speeds. Based on the experience in our lab, we can print 25-micrometer diameter fibers with the following values:2.6 bar, 12 kilovolts, and 700 millimeters per minute.
In all three cases however, fine tuning and optimization will be required to obtain stable results with homogenous diameters. In melt electrospinning, only a perfectly balanced equilibrium between the forces determining the flow of the polymer mass and the forces attracting the jet towards the collector will eventually lead to consistent scaffold morphologies. A well balanced equilibrium is achieved once the flight path of the fiber resembles a catenary curve over a long period of time.
However, three different deviating behaviors are known. First, an unbalanced distribution between delivered mass to the Taylor cone and respective drag forces on the fibers results in a unstable electrified jet. Here the Taylor cone is constantly overfed with polymer and releases its mass in frequent time periods.
Frequently changing angles are visible and result in extremely differing diameters. In order to restabilize the process, decrease the flow rate to minimize polymer delivery and increase the speed and voltage to amplify pulling forces on the fiber. Secondly, the fiber travels down and perpendicularly deposits on the collector.
Hereby the amount of acceleration forces surpasses the polymer supply in the Taylor cone. Slight increases of pressure and lowering the voltage resolves the situation, or altering the collection speed. Thirdly, the fiber lags behind and resembles a straight line under a high angle.
Hereby relatively thick fibers are produced and dragged. This leads to increased inaccuracies of the deposited fibers. A decrease in collection speed will solve the problem.
However, slight increases in the electrical field strength are needed. In melt electrospinning, there are two methods of collection:flat collectors and mandrel collectors. Applying flat collectors refers to the most common method.
090 and 060 degree structures of different sizes are widely reported in literature. The capability of directly depositing molten fibers on the collector also facilitates the production of non-linear structures, such as repetitive circular structures. Tubular architectures can be achieved with cylindrical collectors which rotate as well as traverse parallel to the needle.
Through fine tuning the decode of both the rotational as well as the translational speed, the orientation of the fibers can be modified. Higher rotational speeds than translational speeds lead to radially orientated pores and vice versa. The diameter of the tube depends on the diameter of the implemented mandrel.
A detailed list of different scaffold architectures and their proposed applications in published studies can be found in the manuscript. Due to the highly sensitive electrical field and its specific dependency on the distance between the two electrodes, it is of great importance to regularly level both the collecting stage and the bottom side of the print head. If the distance between the collector and the head is set too close, the electrical field significantly increases until a spark rises between the components.
Turn down the voltage immediately on the electrical control box, or open the door. Before every start of a process, double check all the cable connections and confirm that they are not loose. Any needle damage potentially leads to deformed Taylor cones and thus irregularities within the printing.
Therefore, needles where the tip was bent accidentally must be disposed of. We hope that this video assists in the establishment of a stable electrified jet and that you become convinced, as we are, that melt electrospinning writing will play a significant role in the field of tissue engineering and regenerative medicine.
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