Name: melt electrospinning writing of three-dimensional poly(&#949;-caprolactone) scaffolds with controllable morphologies for tissue engineering applications
Uploaded: 2017-12-23T13:00:00
Description: 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

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. Graph. 29 (4), 1-10 (2010).</li><li>Hochleitner, G., 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=Fibre+pulsing+during+melt+electrospinning+writing.">Fibre pulsing during melt electrospinning writing.</a> BioNanoMaterials. 17 (3-4), 159 (2016).</li><li>Poh, P. S. P., 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=Polylactides+in+additive+biomanufacturing.">Polylactides in additive biomanufacturing.</a> Adv Drug Del Rev. 107, 228-246 (2016).</li><li>Vaquette, C., Cooper-White, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+electrospun+scaffold+pore+size+with+tailored+collectors+for+improved+cell+penetration.">Increasing electrospun scaffold pore size with tailored collectors for improved cell penetration.</a> Acta Biomater. 7 (6), 2544-2557 (2011).</li><li>Thibaudeau, 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=A+tissue-engineered+humanized+xenograft+model+of+human+breast+cancer+metastasis+to+bone.">A tissue-engineered humanized xenograft model of human breast cancer metastasis to bone.</a> Dis Model Mech. 7 (2), 299-309 (2014).</li><li>Holzapfel, B. 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Rapid Commun. 37 (1), 93-99 (2016).</li><li>Wagner, F., 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=A+validated+preclinical+animal+model+for+primary+bone+tumor+research.">A validated preclinical animal model for primary bone tumor research.</a> JBJS. 98 (11), 916-925 (2016).</li><li>Baldwin, J., 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=Periosteum+tissue+engineering+in+an+orthotopic+in+vivo+platform.">Periosteum tissue engineering in an orthotopic in vivo platform.</a> Biomaterials. 121, 193-204 (2017).</li><li>Tourlomousis, F., Chang, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimensional+Metrology+of+Cell-matrix+Interactions+in+3D+Microscale+Fibrous+Substrates.">Dimensional Metrology of Cell-matrix Interactions in 3D Microscale Fibrous Substrates.</a> Procedia CIRP. 65, 32-37 (2017).</li><li>Muerza-Cascante, M. 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=Endosteal-like+extracellular+matrix+expression+on+melt+electrospun+written+scaffolds.">Endosteal-like extracellular matrix expression on melt electrospun written scaffolds.</a> Acta Biomater. 52, 145-158 (2017).</li><li>Delalat, 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=3D+printed+lattices+as+an+activation+and+expansion+platform+for+T+cell+therapy.">3D printed lattices as an activation and expansion platform for T cell therapy.</a> Biomaterials. 140, 58-68 (2017).</li><li>Bas, O., 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=Biofabricated+soft+network+composites+for+cartilage+tissue+engineering.">Biofabricated soft network composites for cartilage tissue engineering.</a> Biofabrication. 9 (2), 025014 (2017).</li><li>Hansske, F., 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=Via+precise+interface+engineering+towards+bioinspired+composites+with+improved+3D+printing+processability+and+mechanical+properties.">Via precise interface engineering towards bioinspired composites with improved 3D printing processability and mechanical properties.</a> J. 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Mater. Eng. 298 (5), 504-520 (2013).</li><li>Wunner, F. M., 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=.">.</a> Comprehensive Biomaterials II. , 217-235 (2017).</li><li>Loh, Q. 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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(&#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.

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.

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Research

JoVE Journal

Bioengineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...