Name: melt electrospinning writing of three-dimensional poly(ε-caprolactone) scaffolds with controllable morphologies for tissue engineering applications
Uploaded: 2017-12-23T13:00:00.000+00:00
Duration: 12 min 28 s
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>Store the PCL pre-loaded syringe at room temperature in a dry and dark environment until it is used.</li>
</ol>
2. Hardware and Software Setup
<ol>
	<li>Attach a 23G flat tipped needle (spinneret) to the syringe and a barrel adapter at the other end to connect the syringe to the air pressure system.</li>
	<li>Place the syringe in the print head and press it down until the spinneret tip stands out 1 mm from the brass part on the bottom side of the head.</li>
	<li>Mount a collector on the stage and clean the surface as well as the print head with 70% (vol/vol) ethanol to remove dust or residual polymer.</li>
	<li>Set the working distance by positioning a 12 mm high object between the spinneret and the collector and lower the print head until the spinneret tip just touches it.</li>
	<li>Adjust the temperature regulators at the electrical box to 82 &#176;C and 65 &#176;C for the spinneret region and the syringe, respectively and power them on to melt the PCL.</li>
	<li>Wait for at least 10 min until the polymer is molten and initiate the air pressure by setting the regulator to 1.8 bar.</li>
	<li>Prepare the G-Code to define the size and shape, inter-filament distance and the number of layers of the scaffold and the collection speed of the process. 
	NOTE: A detailed template for fabricating flat and tubular scaffolds is provided in the discussion chapter (Table 2).</li>
	<li>Double-check manually that all ground cables are connected securely to the enclosure and the wall plug.</li>
	<li>Start the software (e.g., MACH 3) on the computer and upload the prepared G-code.</li>
</ol>
3. Scaffold fabrication
<ol>
	<li>Close the front door of the enclosure, which connects the safety interlock and triggers the high voltage supply to the spinneret. 
	NOTE: Once the door is opened, for example when a print is finished or in case of an emergency, the high voltage drops and the scaffold can be removed safely.</li>
	<li>Increase the high voltage gradually in 0.2 kV steps until a Taylor cone is formed and a fiber is ejected towards the collector (see exemplary Taylor cone in Figure 1D).</li>
	<li>Allow the polymer melt to be extruded on the still collector plate to stabilize the jet without movement for 5 minutes. Remove the pile of material before commencing a new print.</li>
	<li>Use the cursors on the keyboard to move the print head above the point where the G-codes will start.</li>
	<li>Start the G-Code in the software on the computer.</li>
</ol>
4. Fiber Diameter Adjustment
<ol>
	<li>Keep the working distance (12 mm) and the temperature regulators (82 &#176;C and 65 &#176;C for the spinneret region and the syringe, respectively) on a constant level, as described before in steps 2.4 and 2.5. 
	NOTE: A summary of adjusting different diameters is given in Table 3.</li>
	<li>Print fibers with small sized diameters (3-10 &#181;m). Reduce the air pressure level to 0.8 bar, adjust the applied voltage to 8 kV and set the collector speed to 1700 mm/min.</li>
	<li>Print fibers with medium sized diameters (10-20 &#181;m). Adjust the air pressure level to 1.5 bar, set voltage to 11 kV and lower the collection speed to 1200 mm/min.</li>
	<li>Print fibers with large diameters (20-30 &#181;m). Increase the air pressure level to 2.6 bar, alter the applied voltage to 12 kV and decrease the collection speed to 700 mm/min.</li>
</ol>
5. Jet Optimization
<ol>
	<li>Illuminate the jet with a strong LED light from outside the enclosure for improved visibility.</li>
	<li>Observe the behavior of the fiber for 1 minute and adjust the system parameters to optimize the process in small steps, i.e. 0.1 kV for applied voltage, 100 mm/min for collection speed and 0.1 bar for air pressure. 
	NOTE: A summary is given in Table 4.</li>
	<li>Stabilize periodically deflecting behavior by decreasing the air-pressure, increasing the speed and minimizing the voltage until the flight path of the fiber resembles a stable catenary curve for more than 3 minutes.</li>
	<li>Correct the flight path of a lagging behind jet by increasing the voltage, reducing the air pressure and reducing the speed of the collector. Apply those measures until the flight path of the fiber moves back to a catenary curve shape.</li>
	<li>Avoid fibers travelling vertically towards the collector by decreasing the applied voltage, increasing the speed of the collector and increasing the air pressure until the flight path of the jet retains the shape of a catenary curve again.</li>
</ol>
6. Scaffold Collection
<ol>
	<li>Open the door when the print is finished and use the cursor to move the collector plate towards the door for better accessibility.</li>
	<li>Spray the scaffold with ethanol 70% (vol/vol) mix and wait 10 seconds until it visibly detaches from the collector.</li>
	<li>Collect the finished scaffold by grabbing one edge with tweezers and lifting it out of the enclosure.</li>
</ol>
7. Troubleshooting
<ol>
	<li>Decrease the applied voltage or open the door immediately if there is a spark between the spinneret visible or a cracking noise audible.</li>
	<li>Remove all hazardous materials and liquids such as ethanol 70% (vol/vol) from the inside of the enclosure as a fire might ignite in case of potential sparking.</li>
	<li>Program the G-Code accordingly that the spinneret moves away from the area where the scaffold is printed after all layers are done. This avoids material accumulation above the point where the spinneret finally stops.</li>
	<li>Check the spinneret under a magnifier and verify that there is no damage to the spinneret as this will significantly influence the homogeneity of the Taylor cone.</li>
</ol>

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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 ordered structures of biocompatible materials in the micron to nanoscale are required27.
This tutorial aims at generating fundamental knowledge of the operations of MEW by explaining the physical principles and demonstrating action steps to manufacture reproducible scaffolds using this technology.
Since the general principles of MEW are comparable to those of conventional additive manufacturing technologies, i.e. a targeted deposition of extruded material in a layer by layer manner, it is crucial to control the relative movement between the head and the collector. From our experience, we recommend working with MEW devices that keep a fixed head, while the respective movement of the collector is undertaken by the stages (X and Y). A fixated head remains in a stable position and does not generate kinematic forces, which would act on the Taylor cone and potentially lead to disturbances during its creation. In addition, the wiring associated with high voltage and heaters is not subject to sustained repetitive movement. The collector movement is defined by the G-Code, which needs to be uploaded in the software. This code, also known as RS-274, is widely used in the computer-aided manufacturing field to control the pathway of tools. For MEW applications with flat collectors, the G-Code file determines the movement and speed in X and Y direction; for cylindrical collectors or mandrel applications, the G-Code file defines both the translational (X direction) and rotational speeds. Table 2 explains the programming of a G-code in more detail.
Compared to other additive manufacturing technologies, MEW enables the fabrication of fibers with various diameters by the adjustment of the system parameters temperature, collection speed and applied voltage, as described in the protocol.
In order to achieve small fibers (3-10 &#181;m), it is advised to use low pressures, moderate voltages and high collection speeds. Generally, reduced pressure leads to less extruded mass. This is accompanied by a corresponding decrease in the surface area of the jet. Hence, smaller electrostatic forces are required to accelerate the mass of the fiber towards the collector, i.e. lower voltage needs to be applied. Additionally, comparable higher collection speeds lead to enhanced stretching of the fiber, causing an additional reduction of the final fiber diameter.
Increasing the pressure induces more flow of molten polymer and thus, leads to larger fiber diameters (10-20 &#181;m). In this case, greater electrostatic force is required to compensate for the enlarged polymer surface (thicker fibers). In order to obtain a stable polymer jet stream, voltage must be altered and the collection speed should be reduced.
Large fiber diameters (20-30 &#181;m) require enhanced polymer extrusion, i.e. higher air pressure. This provokes relatively thicker fibers and is suggested to be applied in combination with higher voltage to supply sufficient electrostatic force on the fiber. Additionally, reduced collecting speeds induce less fiber stretching. A summary is given in Table 3.
All three cases mentioned above, however, still require fine-tuning and optimization to maintain a stable catenary curve shaped fiber over time, explained in the protocol. In MEW, 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 reaching consistent scaffold morphologies 12,28. Hence, divergences of the pathway of the jet reflect strong deviations of the fiber diameter or inaccurate deposition. From our experience, three different variations in behavior can be obtained.
First, a fiber can pulse, a phenomena initially reported by the Dalton group12. An unbalanced distribution between delivered mass and respective drag-forces on the fibers results in a constantly overfed Taylor cone, which periodically releases accumulated polymer. This causes significant variability in the angles of the pathway and results in differing diameters.
Second, a lagging electrified jet occurs when the speed of the collector is higher than the extrusion speed of the jet. The final deposition of the jet happens far away from the vertical direction of the spinneret, causing a lagging jet stream. The flight path resembles an overemphasized curvature, which also minimizes the dimensions of a printed scaffold.
Third, a buckling electrified jet is caused by the perpendicular impact of the jet onto the collector and manifests, when the collector speed is set slower than the speed at which the jet is flowing out of the spinneret. Applying high voltages can also cause buckling, by producing an excessive acceleration towards the collector and a straight flight path of the fiber. Undesired deposition of loops is observed in this case.
Means to re-stabilize the process are provided in the protocol and shown in Table 4.
From the perspective of scaffold implementation, multiple benefits exist when using PCL and MEW, such as biocompatibility, reproducibility through direct writing, or predesigned customization of the resulting architectures. MEW can be conducted on any conventional laboratory bench, since it uses solvent-free polymer melts, therefore it does not require expensive fume hoods or exhaustive recycling of residual materials29. There is no odor when entering a room containing MEW devices.
Additionally, an achievable high surface to volume ratio within a porous scaffold is of great advantage and makes MEW scaffolds well suited for biological applications30.
In comparison to well-known 3D printing technologies, such as Fused Deposition Modelling31, MEW has limitations in printable heights of ordered structures. The reason is seen in the inherent process of applying electrostatic forces, which traps mobile charge carriers within the deposited fibers. Once the height of scaffolds exceeds approximately 4 mm, it is reported that the sum of the excess charge accumulated within the scaffold acts repelling for upcoming fibers32. Subsequently, in most cases, the resulting top layers are significantly distorted.
Another difference to conventional 3D printing technologies lies in the fact that the deposition of material during the process cannot be interrupted and solely stopping all system parameters eventually holds material extrusion. This represents a design limitation and must be regarded when programming the G-Code. While the jet initiation can be performed mechanically33, G-Code programming needs to consider a continuous direct-writing approach.
Increasing the throughput and process efficiency of MEW also remains a challenge and represents the main reason in our view and others&#39; of why this technology has not been up-scaled to industrial level yet34. First, the MEW process is inherently low in throughput due to the low flow rates and limited collection speeds. Both aspects, however, are essential to ensure the controlled deposition of the jet and reproducibility in the printing. Indeed, the maximum collection speed during the printing process is restricted to the physical boundaries of the material used, i.e. too high speeds would cause breakage of the jet when the drag forces exceed feasible limits. Another strategy to upscale relies on using multi-extruding MEW devices, i.e. machines with multiple printing heads in close distance to each other; however, these multi-heads would cause interferences between the electrical field of each head, and subsequently distort the final fiber deposition35. Needleless melt electrospinning heads have generated a significant number of electrified jets36, although controlling the exact placement of the direct-written fibers could be difficult to achieve. Future developments towards increasing the efficiency of MEW, however, would not only benefit the biomedical community, but also the industries of filtration, textile, or applications for energy2.
Although this tutorial provides guidelines to fabricate customized scaffolds under the proposed parameter settings, it must be noted that minor dependencies on environmental conditions, such as room temperature or humidity exist and might lead to unintended deviations37. The results presented in this tutorial are based on know-how accumulated at the Hutmacher group, conducted in stable environmental conditions within controlled laboratory spaces.
PCL is the most prominent candidate for MEW. From an engineering perspective, its low melting point (60 &#176;C) is beneficial as this does not require the challenging implementation of high temperature heaters (&#62;&#62;100 &#176;C) in close distance to high voltage sources. On a material engineering level, PCL is semi-conductive and provides strong macromolecular cohesion both as a fluid and as a solid. Despite strong mechanical stretching, the viscous material bonds to a certain degree, which results in prominent fiber thinning when increasing collector speed or applied voltage. Conventional melt electrospinning without moving collectors has been reported with different polymers, such as polypropylene, polyethylene or nylon9. The application of direct writing principles however, has predominantly been reported with PCL and some PCL blends with additives to further lower its viscosity38, although there are exceptions39,40. In the future, however, we foresee a wider range of materials processed by MEW. This, in turn, will imply the upgrade of hardware components for this technology, as for example processing polypropylene (melting point at 160&#176;C) alters the current technical requirements of the hardware of MEW devices.
An increasing interest in biocompatible polymeric scaffolds with highly precise and controllable architectures exists; MEW, to date, represents the only technology, which, in comparison to other biomanufacturing techniques, is capable of fabricating ordered architectures in the lower micron range (with exceptions in the sub-micron range41). Within the last years this lead to an exponentially growing amount of patents and publications30. Therefore, tackling technical complexity through implementation of optimized hardware and the establishment of in-process control of MEW is of great importance. This will facilitate the production of scaffolds with tailored architectures for a wide range of applications in the future.

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><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 (&amp;epsilon;-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. 0/90 and 0/60 structures of different sizes are widely reported in literature. Furthermore, the capability of directly depositing molten fibers on the collector also facilitates the production of randomly yet organized structures when a patterned flat collector is used instead of a smooth one14.
Tubular 
There is a great demand for the manufacture of scaffolds with tubular architectures for TE applications. MEW is an effective method to achieve tubular scaffolds with customized porosity by utilizing cylindrical collectors. These rotate along their own axis, while translating along the axis of the mandrel. Through fine tuning of the G-Code, the rotational as well as the translational speed is determined and the orientation of the fibers can be customized. Higher rotational speeds than translational speeds lead to radially orientated pores and vice versa. The total number of layers, distribution and morphology of the porosity will configure the mechanical properties of the scaffold. The inner diameter of the tubular scaffold will be determined by the external diameter of the implemented mandrel.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56289/56289fig1.jpg" /> 
Figure 1: MEW setup. (A) including a PC, the printing unit and the electrical control box (B) the head and the collector (C) the fiber in a perfectly balanced flight phase and (D) a schematic illustration of a Taylor cone. (E) shows a schematic of a printer and lists the five most prevailing system parameters, including &#34;applied voltage&#34; (High Voltage Generator), &#34;temperature&#34; (Temperature Controller), &#34;air pressure&#34; (Pressure Regulator), &#34;working distance&#34; (adjustment via in-house designed movable z-axis) and &#34;collection speed&#34; (X and Y positioning slides). (F) demonstrates the design of the insulation system within the print head via a heat resistant polyamide tape. This prevents arcing between the &#34;heating element 1&#34; and the charged &#34;Brass part&#34;. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56289/56289fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56289/56289fig2.jpg" /> 
Figure 2: Different scaffolds fabricated with a flat collector (A), 0/90 lattice (B) and the same lattice in greater resolution (C). (D) demonstrates a 0/60 structure and (E) a randomly controlled structure. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56289/56289fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56289/56289fig3.jpg" /> 
Figure 3: Showcasing of different tubular scaffolds and one respective representative image from scanning electron microscopy (SEM). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56289/56289fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dermal fibroblast infiltration of poly(&#949;-caprolactone) scaffolds fabricated by melt electrospinning in a direct writing mode (Farrugia et al., 2013)4 ||&#160; FLAT</td>
		</tr>
		<tr>
			<td>Dermal fibroblast seeded PCL MEW scaffolds are evaluated for cell infiltration.</td>
		</tr>
		<tr>
			<td>A tissue-engineered humanized xenograft model of human breast cancer metastasis to bone (Thibaudeau et al., 2014)15 || TUBULAR</td>
		</tr>
		<tr>
			<td>Tubular MEW scaffolds are used to create a viable ectopic &#8216;organ&#8217; bone in a mouse model to study human breast cancer metastasis to bone.</td>
		</tr>
		<tr>
			<td>Species-specific homing mechanisms of human prostate cancer metastasis in tissue engineered bone (Holzapfel et al., 2014)16 || TUBULAR</td>
		</tr>
		<tr>
			<td>MEW scaffolds are used to create a tissue engineered bone for prostate cancer research.</td>
		</tr>
		<tr>
			<td>Enhancing structural integrity of hydrogels by using highly organised melt electrospun fibre constructs (Bas et al., 2015)17 ||&#160; FLAT</td>
		</tr>
		<tr>
			<td>MEW scaffolds with different lay-down patterns and poresizes are used to enhance the mechanical functionality of soft hydrogels.</td>
		</tr>
		<tr>
			<td>Reinforcement of hydrogels using three-dimensionally printed microfibres (Visser et al., 2015)18 ||&#160; FLAT</td>
		</tr>
		<tr>
			<td>Soft gelatin-based hydrogels are reinforced with MEW PCL scaffolds.</td>
		</tr>
		<tr>
			<td>Melt electrospinning onto cylinders: effects of rotational velocity and collector diameter on morphology of tubular structures (Jungst et al., 2015)6 || TUBULAR</td>
		</tr>
		<tr>
			<td>The influence of the translational and rotational speeds on the final morphology of tubular MEW scaffolds are systematically investigated.</td>
		</tr>
		<tr>
			<td>Hierarchically structured porous poly(2-oxazoline) hydrogels (Haigh et al., 2016)19 || FLAT</td>
		</tr>
		<tr>
			<td>MEW scaffolds are used as a sacrificial template to create a hierarchical 3D porosity network within a hydrogel.</td>
		</tr>
		<tr>
			<td>A validated preclinical animal model for primary bone tumor research (Wagner et al., 2016)20 || TUBULAR</td>
		</tr>
		<tr>
			<td>MEW scaffolds are used to create humanized tissue engineered constructs for preclinical research on primary bone tumors.</td>
		</tr>
		<tr>
			<td>Periosteum tissue engineering in an orthotopic&#160;in&#160;vivo&#160;platform (Baldwin et al., 2017) 21 || TUBULAR</td>
		</tr>
		<tr>
			<td>A multiphasic scaffold consisting of a MEW mesh and a hydrogel is developed for periosteum tissue regeneration applications.</td>
		</tr>
		<tr>
			<td>Dimensional metrology of cell-matrix interactions in 3D microscale fibrous substrates (Tourlomousis and Chang. 2017)22 || FLAT</td>
		</tr>
		<tr>
			<td>Cell-matrix interactions are investigated on MEW scaffolds with different architectures.</td>
		</tr>
		<tr>
			<td>Endosteal-like extracellular matrix expression on&#160;melt&#160;electrospun written scaffold (Muerza-Cascante et al., 2017)23 || FLAT</td>
		</tr>
		<tr>
			<td>MEW PCL scaffolds are used to develop an endosteal bone-like tissue that promotes the growth of primary human haematopoietic stem cells.&#160;</td>
		</tr>
		<tr>
			<td>3D printed lattices as an activation and expansion platform for T cell therapy (Delalat et al., 2017)24 || FLAT</td>
		</tr>
		<tr>
			<td>Scaffolds with different fiber spacing (200 &#181;m, 500 &#181;m and 1000 &#181;m) are surface functionalised and seeded with T cells for expansion.</td>
		</tr>
		<tr>
			<td>Biofabricated soft network composites for cartilage tissue engineering (Bas et al., 2017)25 || FLAT</td>
		</tr>
		<tr>
			<td>Biomimetic soft network composites consisting of a hydrogel matrix and reinforcing MEW meshes designed for articular cartilage repair are reported.</td>
		</tr>
		<tr>
			<td>Via precise interface engineering towards bioinspired composites with improved 3D printing processability and mechanical properties (Hansske et al., 2017)26 || FLAT</td>
		</tr>
		<tr>
			<td>Magnesium fluoride nanoparticle reinforced PCL scaffolds fabricated by means of MEW are designed and developed for bone tissue engineering applications.</td>
		</tr>
	</tbody>
</table>
Table 1: References to a list of studies, in which MEW scaffolds were fabricated and used for biological applications. The list provides results of implemented flat as well as tubular scaffolds.
<img alt="Table 2" src="/files/ftp_upload/56289/56289tbl2.jpg" /> 
Table 2: Explanation of programming a G-Code for flat and tubular scaffolds, using a text file (.txt) to be uploaded in the software. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56289/56289tbl2large.jpg" target="_blank">Please click here to view a larger version of this table.</a>
<img alt="Table 3" src="/files/ftp_upload/56289/56289tbl3.jpg" /> 
Table 3: Representative values of the parameters air pressure, voltage and collection speed (temperature and collection distance constant) to reach three different diameter ranges (small, medium and large). The red arrows propose exact values within the respective categories to reach the fiber diameters. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56289/56289tbl3large.jpg" target="_blank">Please click here to view a larger version of this table.</a>
<img alt="Table 4" src="/files/ftp_upload/56289/56289tbl4.jpg" /> 
Table 4: Schematic illustration of the different cases and real images of possible fiber deposition at MEW as well as means to optimize. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56289/56289tbl4large.jpg" target="_blank">Please click here to view a larger version of this table.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

15.1K Views.  Queensland University of Technology (QUT). Dit protocol fungeert als een uitgebreide richtsnoer te fabriceren steigers via electrospinning met polymeer in een directe schrijven-modus smelt. Wij systematisch overzicht van het proces en de juiste parameterinstellingen definiëren voor het bereiken van gerichte steiger platforms.

Video: Melt Electrospinning Writing of Three-dimensional Poly(ε-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45-45-10, 55-35-10, and 65-25-10 PCL-ELAS-COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration. Whole grafts and individual layers were analyzed using uniaxial tensile testing, dynamic compliance, suture retention, and burst strength. Compliance results revealed that changes to the middle/medial layer changed overall graft behavior with whole graft compliance values ranging from 0.8 - 2.8 % / 100 mmHg, while uniaxial results demonstrated an average modulus range of 2.0 - 11.8 MPa. Both modulus and compliance data displayed values within the range of native artery. Mathematical modeling was implemented to show how changes in layer stiffness affect the overall circumferential wall stress, and as a design aid to achieve the best mechanical combination of materials. Overall, the results indicated that a graft can be designed to mimic a tri-layered structure by altering layer properties.

The aim of this study was to mimic the native three layered architecture of the arterial wall. To accomplish this, electrospinning was employed with the use of a 3-1 (input-output) nozzle and blends of polycaprolactone, elastin, and collagen.

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease control of composition, structure, and diameter of fibers. However, despite these advantages, traditional electrospun nanofiber scaffolds come with limitations including disorganized nanofiber orientation, low porosity, small pore size, and mainly two-dimensional mats. As such, there is a great need for developing a new process for fabricating electrospun nanofiber scaffolds that can overcome the above limitations. Herein, a novel and simple method is outlined. A traditional 2D nanofiber mat is transformed into a 3D scaffold with desired thickness, gap distance, porosity, and nanotopographic cues to allow for cell seeding and proliferation through the depressurization of subcritical CO2 fluid. In addition to providing a scaffold for tissue regeneration to occur, this method also provides the opportunity to encapsulate bioactive molecules such as antimicrobial peptides for local drug delivery. The CO2 expanded nanofiber scaffolds hold great potential in tissue regeneration, wound healing, 3D tissue modeling, and topical drug delivery.

This article demonstrates the technique of expanding a traditional, two-dimension (2D) electrospun nanofiber mat into a three-dimension (3D) scaffold through the depressurization of subcritical CO2 fluid. These augmented scaffolds are 3D, closely mimic cellular nanotopographic cues, and preserve the functions of biologic molecules encapsulated within the nanofibers.

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease ...

Genetics

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

Biology

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Controlling Electrospun Polymer Morphology for Tissue Engineering Demonstrated Using hepG2 Cell Line

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of electrospun polymers is regarded as a structural imitation of the extracellular matrix (ECM). Hence, electrospun fibers fabricated from biocompatible polymers have been widely investigated by tissue engineering researchers for their potential role as an artificial ECM for guiding tissue growth both in vitro and in vivo. All cells are acutely sensitive to their mechanical environment. This has been demonstrated by the discovery of multiple mechanotransduction pathways intrinsically linked to the cytoskeletal actin filaments. The cytoskeleton acts as a mechanical sensor that can direct the functionality and differentiation of the host cell depending on the stiffness and morphology of its substrate. Electrospun fibers can be tuned both in terms of fiber size and morphology to easily modulate the mechanical environment within a fibrous polymer scaffold. Here, methods for electrospinning polycaprolactone (PCL) for three distinct morphologies at two different fiber diameters are described. The morphological fiber categories consist of randomly oriented fibers, aligned fibers, and porous cryogenically spun fibers, with 1 &#181;m and 5 &#181;m diameters. The methods detailed within this study are proposed as a platform for investigating the effect of electrospun fiber architecture on tissue generation. Understanding these effects will allow researchers to optimize the mechanical properties of electrospun fibers and demonstrate the potential of this technology more thoroughly.

This method provides the means to test different polycaprolactone fiber morphologies and topographies for the purpose of tissue engineering. Small and large fibers are fabricated with random orientations, aligned orientations, and also porous cryogenically electrospun structures and used as platforms for cell culture.

Melt Electrospinning Writing of Three-dimensional Poly(ε-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

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