This work has been financially supported by the Cooperative Research Centre CRC for Cell Therapy Manufacturing, the Australian Research Council ARC Centre in Additive Biomanufacturing and the Institute for Advanced Study of the Technical University of Munich. This research was conducted by the Australian Research Council Industrial Transformation Training Centre in Additive Biomanufacturing http://www.additivebiomanufacturing.org (IC160100026). Please visit the site for articles, books, television or radio programs, electronic media, or any other literary works related to the Project. Further, the authors gratefully acknowledge Maria Flandes Iparraguirre for support in filming, Philip Hubbard for the voice over and Luise Grossmann for filming and editing.

1. Material Preparation
<ol>
	<li>Fill 2 g of PCL in a 3 mL plastic syringe with a funnel and insert a piston into the open end.</li>
	<li>Place the syringe in a preheated oven at 65 &#176;C for 8 h. Point the tip upwards to allow the air bubbles to aggregate close to the opening.</li>
	<li>Push the piston with a thin object to release the trapped air within the molten material.</li>
	<li>Let it cool down to room temperature, which is achieved when the polymer is not transparent anymore after 10 minutes.</li>
	<li>Stor...

<ol>
	<li>Muerza-Cascante, M. L., Haylock, D., Hutmacher, D. W., Dalton, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melt+Electrospinning+and+Its+Technologization+in+Tissue+Engineering.">Melt Electrospinning and Its Technologization in Tissue Engineering.</a> Tissue Eng Part B Rev. 21 (2), 187-202 (2015).</li><li>Brown, T. D., Dalton, P. D., Hutmacher, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melt+electrospinning+today:+An+opportune+time+for+an+emerging+polymer+process.">Melt electrospinning today: An opportune time for an emerging polymer process.</a> Prog. in pol Sci. , (2015).</li><li>Brown, T. D., Dalton, P. D., Hutmacher, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+writing+by+way+of+melt+electrospinning.">Direct writing by way of melt electrospinning.</a> Adv Mater. 23 (47), 5651-5657 (2011).</li><li>Farrugia, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dermal+fibroblast+infiltration+of+poly(&#949;-caprolactone)+scaffolds+fabricated+by+melt+electrospinning+in+a+direct+writing+mode.">Dermal fibroblast infiltration of poly(&#949;-caprolactone) scaffolds fabricated by melt electrospinning in a direct writing mode.</a> Biofabrication. 5 (2), 025001 (2013).</li><li>Brown, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melt+electrospinning+of+poly+(&#949;-caprolactone)+scaffolds:+Phenomenological+observations+associated+with+collection+and+direct+writing.">Melt electrospinning of poly (&#949;-caprolactone) scaffolds: Phenomenological observations associated with collection and direct writing.</a> Mater. Sci. Eng. C. 45, 698-708 (2014).</li><li>Jungst, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melt+electrospinning+onto+cylinders:+effects+of+rotational+velocity+and+collector+diameter+on+morphology+of+tubular+structures.">Melt electrospinning onto cylinders: effects of rotational velocity and collector diameter on morphology of tubular structures.</a> Polym Int. 64 (9), 1086-1095 (2015).</li><li>Brown, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+Fabrication+of+Tubular+Scaffolds+via+Direct+Writing+in+a+Melt+Electrospinning+Mode.">Design and Fabrication of Tubular Scaffolds via Direct Writing in a Melt Electrospinning Mode.</a> Biointerphases. 7 (1), 1-16 (2012).</li><li>Woodruff, M. A., Hutmacher, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+return+of+a+forgotten+polymer-Polycaprolactone+in+the+21st+century.">The return of a forgotten polymer-Polycaprolactone in the 21st century.</a> Prog. in pol Sci. 35 (10), 1217-1256 (2010).</li><li>Hutmacher, D. W., Dalton, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melt+electrospinning.">Melt electrospinning.</a> Chem Asian J. 6 (1), 44-56 (2011).</li><li>Wei, C., Gang, T. Q., Chen, L. J., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+condition+for+the+transformation+from+Taylor+cone+to+cone-jet.">Critical condition for the transformation from Taylor cone to cone-jet.</a> Chin. Phys. B. 23 (6), 064702 (2014).</li><li>Mikl, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrete+viscous+threads.">Discrete viscous threads.</a> ACM Trans. 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The authors have nothing to disclose.

Integrating AM in order to find innovative solutions for the challenges in the medical field presents a new paradigm for the 21st century. The so-called field of &#34;Bio-fabrication&#34; is on the rise and innovations in fabrication technologies enable the production of highly sophisticated architectures for TE applications. The electrospinning of polymer melts in a direct writing mode (here MEW) is seen as one of the most promising manufacturing candidates to comply with the needs of the TE community, where ...

The manufacture of three-dimensional (3D) biocompatible structures for cells is one of the key contributions of additive biomanufacturing to tissue engineering (TE), aiming to restore tissues by applying customized biomaterials, cells, biochemical factors, or a combination of them. Therefore, the main requirements of scaffolds for TE applications include: manufacturability from biocompatible materials, controllable morphological properties for targeted cell invasion and optimized surface properties for enhanced cell interaction1.
MEW is a solvent-free manufacturing technique that combines the principles of additive manufacturing (often called 3D printing) and electrospinning for the production of polymeric meshes with highly ordered ultrathin fiber morphologies2. It is a direct writing approach and accurately deposits fibers according to preprogrammed codes3, referred to as G-Codes. Melt electrospun constructs are currently prepared using a flat4,5 or a mandrel6,7 collector to fabricate porous flat and tubular scaffolds, respectively.
This technique offers significant benefits to the TE and regenerative medicine (RM) community due to the possibility to directly print medical-grade polymers, such as poly(&#949;-caprolactone) (PCL), which presents excellent biocompatibility8. Other advantages are the possibility to customize the size and distribution of the porosity, by depositing the fibers in a highly-organized manner to fabricate scaffolds of high surface-to-volume ratios. Before MEW can be performed, the polymer first requires the application of heat9. Once in a fluid state, an applied air pressure forces it to flow out through a metallic spinneret that is connected to a high voltage source. The force balance between the surface tension and the attraction of the electrostatically charged droplet to the grounded collector leads to the formation of a Taylor cone followed by the ejection of a jet10.
Images and a schematic drawing of the in-house build MEW device used for this protocol are shown in Figure 1. It additionally demonstrates the principles of using insulating tape to avoid electrical discharge between the heating elements and the electrically charged brass part surrounding the spinneret. Insufficient insulation would lead to internal damage of the implemented hardware.
Depending on the adjustment of the three system parameters (temperature, collection speed and air pressure), MEW enables the fabrication of fibers with different diameters, explained in the discussion section. In most cases, however, fine-tuning and optimization of the jet will be required before a stable jet will be ejected. The visualization of the electrified travelling jet is an effective way to verify the consistency and homogeneity of the process. In an ideal case, the flight path resembles a catenary curve acquired as a result of a force balance controlled by the system parameters11. Further, the micro- and macro-structure of the scaffolds is dependent on the flight path of the polymer jet12. A detailed table of different deflection behaviors and measures for optimization is given in the discussion section.
In the present study, we present a protocol that describes the fabrication steps for the manufacture of highly controlled fibrous scaffolds using MEW technology. In this work, medical grade PCL (molecular weight 95-140 kg/mol) was used, as this medical grade PCL has improved purity over technical grade, and its mechanical and processing properties are excellent for MEW. Broad melt processing range of PCL originates from its low melting point (60 &#176;C) and high thermal stability. Moreover, PCL is a slow-rate biodegradable polymer, which makes it an excellent material for many tissue engineering applications13.
For this study, the temperature and collector distance will be kept constant (65 &#176;C and 82 &#176;C for the syringe and spinneret temperatures (respectively) and 12 mm for the collector distance); applied voltage, collector speed and air pressure, however, will be varied to fabricate fibers with targeted diameters. A detailed list of published studies using MEW scaffolds is provided in the results section and reveals different applications for the fields of TE and RM (Table 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plastic syringe</td>
			<td>Nordson Australia Pty Ltd</td>
			<td>7012072</td>
			<td>EFD BARREL O 3mL Clear 50</td>
		</tr>
		<tr>
			<td>Medical grade Poly (&#949;-caprolactone) (mPCL)</td>
			<td>Corbion Purac, The Netherlands</td>
			<td>PURASORB PC12</td>
			<td></td>
		</tr>
		<tr>
			<td>23 GA needle</td>
			<td>Nordson Australia Pty Ltd</td>
			<td>7018302</td>
			<td>#23GP .013 X .25 ORANGE 50 PC</td>
		</tr>
		<tr>
			<td>Plunger</td>
			<td>Nordson Australia Pty Ltd</td>
			<td>7012166</td>
			<td>PISTON O 3mL WH WIPER 50</td>
		</tr>
		<tr>
			<td>Pressure adapter</td>
			<td>Nordson Australia Pty Ltd</td>
			<td>7012059</td>
			<td>ADAPTER ASM O 3mL BL 1.8M</td>
		</tr>
		<tr>
			<td>Aluminium collector</td>
			<td>Action Aluminium, Australia</td>
			<td>SHP2</td>
			<td>Sheet 5005 H34</td>
		</tr>
		<tr>
			<td>Acrylic glass</td>
			<td>Mulford Plastics Pty Ltd</td>
			<td>ACC6-13094</td>
			<td></td>
		</tr>
		<tr>
			<td>Mach 3 software</td>
			<td>Art Soft</td>
			<td></td>
			<td>Purchased online</td>
		</tr>
		<tr>
			<td>Safety switch interlock</td>
			<td>RS components Pty Ltd</td>
			<td>12621330</td>
			<td></td>
		</tr>
		<tr>
			<td>High voltage generator</td>
			<td>EMCO High Voltage Co.</td>
			<td>DX250R</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>WATLOW</td>
			<td>PM9R1FJ</td>
			<td></td>
		</tr>
		<tr>
			<td>X and Y positioning slide</td>
			<td>VELMEX Inc.</td>
			<td>XN-10-0020-M011</td>
			<td></td>
		</tr>
	</tbody>
</table>

melt electrospinning writing of three-dimensional poly(&#949;-caprolactone) scaffolds with controllable morphologies for tissue engineering applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Two different methods of collection are commonly used in MEW, which are flat collection and mandrel collection. The resulting architectures depend on the programming of the G-Code (Table 2), which is executed by the software.
Flat collection 
Applying flat collectors refers to the most common method and facilitates the direct deposition of material referring to the pre-programmed G-code. 0/...

Watch this Scientific Journal Video about Melt Electrospinning Writing of Three-dimensional PolyÎµ-caprolactone Scaffolds with Controllable Morphologies for Tissue Engineering Applications at JoVE.c

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

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.

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

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.

melt-electrospinning-writing-three-dimensional-poly-caprolactone

Research

JoVE Journal

Bioengineering

15.0K ビュー.  Queensland University of Technology (QUT). 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 requi...

ビデオ: Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications