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 PolyÃŽÂµ-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 PolyÃŽÂµ-caprolactone Scaffolds with Controllable Morphologies for Tissue Engineering Applications at JoVE.c

Melt Electrospinning Writing of Three-dimensional PolyÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Âµ-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 ...

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 ...

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 ...

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 ...

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 ...

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, ...

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 ...

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 ...

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, ...

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

Fabrication of Inverted Colloidal Crystal Polyethylene 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 ...

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 ...