There are many polymer processing technologies available to manufacture scaffolds for tissue engineering (TE) , , . Some, such as porogen leaching  or thermally induced phase separation , were initially developed decades ago, while more recent additive manufacturing (AM) principles have also been used to fabricate scaffolds , , . In contrast to other processing techniques, AM can be used for superior shaping of the printed scaffolds by using a computer-aided process to design and control the porosity, mesh-width, fibre or strut diameter, and wall thickness in three dimensions , . This processing flexibility can be used to tailor structural gradient or zonal constructs and is therefore particularly interesting for TE approaches and scaffold manufacturing , .
Recently, melt electrospinning writing (MEW) has been described as an AM technology for scaffold-based TE applications , . MEW is based on electrohydrodynamic direct writing, where a molten jet is stabilised at very low flow rates with an accelerating voltage. This electric stabilisation of a jet  (i.e. with no droplet formation) at low flow rates provides a predictable jet path that allows continuous direct writing and layer by layer assembly. This results in fibres, that are substantially smaller than those produced by fused deposition modelling (FDM) , , typically 5–40 μm , ,  and as small as 800 nm . Importantly, the fibres can be accurately deposited upon each other, so that three-dimensional (3D) structures can be designed and built to millimetre thicknesses .
While MEW is a relatively new processing technology, there is an increasing body of evidence suggesting that there are multiple advantages to using it for fabricating scaffolds , , , , . MEW is solvent-free – therefore the need to print within a fume hood or to use special equipment to remove volatile chemicals is not required, and solvent accumulation in fibres does not occur. Another benefit of MEW is that small fibre diameters lead to flexible constructs that enable even relatively rigid polymers to be fabricated as soft, compliant structures , , . So far, melt electrospinning has been used to process the following medical polymers: poly(ε-caprolactone) (PCL) , , , , , , , poly(lactide-co-glycolide) , poly(lactic acid) , PCL-block-poly(ethylene glycol) , , , , , poly(lactide-co-caprolactone-co-acryloyl carbonate) , polypropylene , , poly(methyl methacrylate) , and poly(2-ethyl-2-oxazoline) (PEtOx) .
When comparing MEW to standard solution electrospinning, it is important to note that the polymer melts used have significantly different physical properties than a typical polymer solution. Both the fluid conductivity and viscosity vary and this influences fabrication aspects such as the final diameter , electrified jet path  and Coulomb interaction of previously deposited fibres. One result of this is that direct writing from melts can be performed at a relatively large range of distances between the spinneret and the collector, from hundreds of microns up to 10 cm , . In case of melt electrospinning the instrumental parameters, including the electrification of the jet, must be adjusted to each other in order to generate a stable process leading to a high quality fibre deposition.
For MEW, when the mass flow rates from the spinneret onto the collector are not adjusted, the phenomenon of “pulsing” occurs. Herein, “pulsing” is defined as the undesired sectional oscillation of the fibre diameter. This is a serious polymer processing issue to overcome for MEW, since it affects the quality of the printed structure. Pulsing also influences the fibre placement when there is a change in direction of the collector stage (i.e. turning). Therefore, key scaffold characteristics such as stiffness, fibre spacing or specific surface area of the printed constructs will not be constant when fibre pulsing occurs (Figure S1). In this research paper, we will describe certain conditions where the fibre pulsing instability manifests and how to eradicate this phenomenon from MEW scaffolds by varying the instrumental parameters: acceleration voltage and feeding pressure. The samples were made of PCL, a long-term hydrolytically degradable polymer that is increasingly used in TE research  and the clinic . Scanning electron microscopy (SEM) analysis of fibres was done using both stable and pulsing conditions – and produced grids which are the unit repeat structure for TE scaffolds made by MEW , . Furthermore, we present a simple approach to visually detect and overcome pulsing that can be used as a guideline for manufacturing high quality TE scaffolds.
PCL was sourced from Corbion Inc. (PURASORB PC 12, Lot# 1412000249, 03/2015, Gorinchem, Netherlands) and used throughout this study. To avoid polymer degradation during storage, the PCL was repacked into 50 mL Falcon tubes, under a N2 atmosphere in a glove box to ensure proper storage conditions. Once repacked, the polymer was stored at –80 °C.
Experiments were performed with a custom-built MEW device based on a commercially available CNC platform. Within an aluminium frame box (68×60×80 cm) with polycarbonate windows (Figure 1), an x-y slide system (Bosch Rexroth AG, Lohr am Main, Germany) was mounted on an aluminium plate and connected to programmable logic controllers (Bosch Rexroth AG, Lohr am Main, Germany). An electrical heater was utilised that contained disposable syringes (Nordson EFD Deutschland GmbH, Pforzheim, Germany) filled with approximately 0.5 mL–1 mL PCL and linked to a digitally controlled air pressure system (Bosch Rexroth AG, Lohr am Main, Germany). A 22 G spinneret (Nordson EFD Deutschland GmbH, Pforzheim, Germany), with a length of 12.5 mm protruded 0.5 mm out of the high voltage (HV) electrode supplied by a positive HV source (LNC 10000-5 pos, Heinzinger Electronic GmbH, Rosenheim, Germany). The negative HV source (LNC 10000-5 neg, Heinzinger Electronic GmbH, Rosenheim, Germany) was connected to a 200×175 mm stainless steel platform (with the collector placed on this platform), attached to the x-y slide system.
Melt electrospinning was performed at 21±2 °C with a relative humidity of 40±10%. The polymer melt temperature for standard conditions was 73±1 °C unless specifically stated otherwise and the polymer was used between 1 and 5 days. Fibre pulsing regimes were investigated at different flow rate conditions using a 22 G spinneret at 0.2, 0.5, 1.0, and 2.0 bar. These experiments were performed with 6.0 kV acceleration voltage. Acceleration voltage refers here to a difference between negative voltage on the collector, which was always set to –1.5 kV and a variable positive spinneret voltage. In addition, the effect of the acceleration voltage at 4.50, 5.25 and 6.00 kV was investigated at 1.0 bar pressure. The distance between the spinneret tip and the collector surface (spinning length) was maintained at 4 mm for all experiments, if not specifically stated otherwise.
Digital signals in the form of G-codes were generated to enable the MEW device to print different lines of fibres in alternating 0° and 90° directions to form grids, as well as fibre line arrays for diameter quantification. Printing was done on a collector consisting of glass slides (26×50 mm) for fibre diameter measurements and stainless steel plates for scaffold processing (50×70 mm).
Electrified PCL jet images were obtained using an AxioCam 105 colour digital camera (Carl Zeiss Microscopy, Göttingen, Germany), mounted on a 75–300 mm lens (Kenko Tokina Co., Ltd., Tokyo, Japan) outside the polycarbonate housing aimed at the spinneret. In order to analyse printed fibre lines and grid structures a stereomicroscope (Discovery V20, Carl Zeiss Microscopy GmbH, Göttingen, Germany) was used. SEM of the printed grids and scaffolds was performed using a Crossbeam 340 SEM (Carl Zeiss Microscopy, Göttingen, Germany) equipped with a Zeiss Gemini column. Quantitative SEM imaging of grids and line arrays was performed with samples as printed, with no coating applied to the surfaces, while qualitative images of scaffolds were sputter coated with platinum. SEM images were taken of specific regions of the printed grids, line arrays and edges to investigate the morphology of the printed fibres. SEM analysis was performed to determine diameters of the fibres by systematically measuring fibre diameters for the printed line arrays. Furthermore, for each varied parameter three repeats of the line array samples were analysed with 20 alternate fibres measured on each sample. The edges of both the grids and line arrays provided information related to the turn points.
Results and discussion
In this study, we assume a conservation of polymer masses with m1+m2+m3=constant, where m1 is the polymer melt in the spinneret (including reservoir), m2 the mass of the jet and m3 the mass of the deposited fibres. Figure 2 illustrates the corresponding mass flows of the MEW process schematically. During every single printing experiment, the polymer melt mass flow dm1/dt to the spinneret was kept constant (d2m1/dt=0). To investigate the oscillation of the resulting fibre mass flow dm3/dt we characterised deposited fibre diameters manufactured at constant collector speeds. This procedure enables an evaluation of a time-dependent fluctuation of the jet with dm2/dt. During the experiments no jet breaks were observed, even under pulsing or long beading conditions.
Effect of collector speed on fibre morphology
In MEW, straight fibres are printed when the computer-controlled collector movement speed vcol is greater than the ultimate jet speed, termed the critical translation speed (CTS) as described previously  (Figure 3A). When processing below this CTS value, non-linear fluid patterns due to jet buckling can be observed (Figure 3B–D). These types of patterns are already described for falling viscous liquids (syrup, silicone oil) under non-electrohydrodynamic conditions , , . Such patterns have been described as: sinusoidal meanders (Figure 3B), having “sidekicks” or translated coiling (Figure 3C), and “figure of eight” loops (Figure 3D). The difference is that here, the molten PCL fibre solidifies due to cooling  while the syrup and silicone fluids coalesce and lose their shape. The uniform patterns shown for PCL in Figure 3 demonstrate that the molten electrified jet is stable with time, since any slight change in the diameter would affect the deposition behaviour.
At collector speeds above the CTS, the fibre diameter can be reduced by a high ratio of vcol/CTS (Figure 4A–C) due to mechanical stretching. Another effect of increasing collector speed is a growing lag of the jet. The first contact point of the fibre on the collector lags behind the location of the spinneret, as the collector moves and drags the electrified molten jet. This is especially visible on the deposited fibre at turning points (Figure 4A). The magnitude of this lag defines the discrepancy between the programmed collector movement and the deposited fibre path. Figure 4D–G shows the electrified molten jet being drawn along the surface of the collector at different speeds above the CTS.
Different types of pulsing can be observed and are related to the duration of printing, Figure 5 gives a general explanation of the pulsing role in the jet geometry. When the electrified jet diameter varies from a thick to a thin profile (and vice versa), there is a visible change of the variable lag. Changes in the variable lag have a particular impact on fibre placement when turning during writing and therefore affects the scaffold quality after printing multiple layers.
Accordingly, we observed a temporary jet oscillation initiated by changes of instrumental parameters (e.g. feeding pressure) which seemed to shift the dynamic process and thus force balance to another equilibrium level. This shift in balance of parameters can cause a “temporary pulsing” instability, which leads to an initial disequilibrium of mass flows that equilibrates to a homogenous deposition profile (Figure 5B). We observed that such temporary jet oscillations can last several minutes before stabilising of the jet, as long as the combination of instrumental parameters is adjusted for stable printing.
The variable lag of the jet is of particular interest to the controlled placement of fibres during MEW. A balance of the instrumental parameters with respect to the influence of the jet speed on jet thickness is crucial in obtaining controlled scaffold manufacture. As shown in Figure 5A, a larger mass flow of the polymer melt through the spinneret (dm1/dt) not only led to thicker fibres, but also to a lower CTS . As a result, the pulsing phenomenon can be observed as a wave-like structural feature at the turn edge (Figure 5B), given that MEW operates above the CTS throughout. This is a simple visual tool to evaluate if optimization of instrumental parameters is still required. The effect of changes in the jet profile due to pulsing can be compared to the programmed path of a regular deposition pattern. In case where the new equilibrium state cannot be achieved, for instance due to insufficient electrical field strength, wave-like edge is preserved throughout the constructs (Figure S1). This denotes such processing phenomenon as continuous pulsing.
The instrumental parameters (22 G; 5.25 kV, 4 mm collector distance and 1.0 bar) for the demonstration of continuous pulsing were selected as they provided excessive flow rate conditions (dm1/dt) that resulted in continuous oscillation of fibre diameters in the following experiments. We previously described this effect as “fibre pulsing” for the MEW processing of PEtOx . To demonstrate that fibre pulsing is also applicable to PCL, we investigated this phenomenon further. As illustrated in Figure 2, the polymer melt mass flow dm1/dt through the spinneret is a key component of fibre pulsing and is largely governed by the feeding pressure, melt viscosity and spinneret diameter/length, according to the Hagen-Poiseuille equation , . The described correlation in this formula is valid for viscous, Newtonian fluids and laminar flow conditions and therefore does not fully apply for polymer melts, but nonetheless the equation can be helpful to assess the influence of instrumental parameters during MEW. When the spinneret size is constant during processing, the feeding pressure can be used to influence dm1/dt. Moreover, the feeding pressure seems to be the most suitable parameter for changing dm1/dt due to its broad adjustable range (here we used one order of magnitude: 0.2–2.0 bar) without additional consequences or limitations for the printing process beside pulsing phenomena.
Increased pressure can be compensated by greater magnitude of forces, stretching the jet, induced by an electrical field. Nevertheless, an excessive voltage leads to arcing and a breakdown of the electrical field. In our experience this resulted in a narrow usable voltage range at a fixed collector distance, while the acceleration voltage needs to be at a certain minimum value in order to form a Taylor Cone , Figure 7 shows that the increase in acceleration voltage from 5.25 kV to 6.00 kV resulted in more homogeneously printed fibres with 15.56±1.16 μm to 15.50±0.46 μm, respectively. According to the printing conditions with d2m1/dt=0, a stronger electrical field tended to stabilise the jet mass flow dm2/dt at the studied conditions. Hence, parameters which define forces affecting dm1/dt and dm2/dt must be balanced in order to avoid continuous pulsing with d2m2/dt≠0. Under pulsing conditions, the resulting force value which leads to jet stretching (as a function of Coulomb interaction, mechanical stretching, surface tension and counteracting restoring forces of the viscoelastic polymer etc.) cannot be constant during pulsing.
Long bead defects
For the samples produced at 2.0 bar (Figure 6A, B1–B2) and 4.5 kV (Figure 7A, D1–D2) the fibre diameter oscillation was even more evident than continuous pulsing. This phenomenon was termed here “long beading” which describes the shape of the printed instability. While both pulsing and long beading indicate an oscillation of the jet profile and can be observed at the edges of a printed construct, long beads can be characterised as outliers in the sorted function of fibre diameters at sufficient number of measurement points (n=60). In the case of 1.0 bar (Figure 6C1–C2), 0.5 bar (Figure 6D1–D2), and 0.2 bar (Figure 6E1–E2), no remarkable fibre oscillation of dm3/dt occurred, and homogeneous fibre diameters (15.40±0.60, 11.04±0.41, and 7.36±0.35 μm respectively, Table 1) (average±SD) could be observed (Figure 8). This is expressed by small deviation with median and averages of measured fibre diameters being very similar. In contrast, the 2.0 bar samples contained long beads and had comparatively large deviations recorded for fibre diameter measurements (19.4±8.4 μm) with the measured diameters ranging from 8.0 μm up to 46.4 μm (Figure 8B). A visualisation of a representative long bead is provided in the supplementary information (Figure S2).
The fibre diameter for both experimental series (pressure and voltage) can be analysed with the graphical application of the average in combination with the median. This seems to be a simple tool to analyse the influence of long beading during MEW, since a homogenous jet diameter results in a narrow and symmetrical distribution function (Figure 8) with a ratio of median/average (m/a) close to 1.0. In contrast, long beading as a result of high pressures (2.0 bar) or low voltages (4.50 kV) was expressed by a difference of 14% (m/a=0.86) and 25% (m/a=0.75), respectively. Thus, the asymmetry of the distribution functions indicates the appearance and the intensity of long beading.
Studying the mass flows and visualisation of the process can provide a better understanding of process stability during MEW. A decrease in acceleration voltage at constant dm1/dt led to time-dependant oscillation of jet and fibre mass flows with d2m2/dt≠0 and thus d2m3/dt≠0. A similar effect can be observed when the feeding pressure is increased under constant electrical field strength. Then an increased dm1/dt led to an excessive material extrusion, initiating the time dependant fibre oscillation of d2m3/dt≠0 (continuous pulsing or long beading). The electrical field induces a force acting on the molten polymer jet leading to melt acceleration and therefore influences the stability of dm2/dt (electrified jet mass). If the acceleration voltage was not sufficient or the feeding pressure too high, then continuous pulsing or long beading occurred. We hypothesise that the capacity of the electrical field to accelerate the mass to the collector was exceeded by a high extrusion rate through the spinneret. After a certain time, forces acting on the accumulated polymer at the spinneret tip overcome the mechanical resistance of the material. Then, the melt is drawn to the collector in the form of a thicker jet, preserving the Taylor cone (pulsing) or as an elongated drop (long beading). Afterwards the jet gets thinner and CTS increases. This behaviour in terms of fibre diameter change over time is illustrated in Figure 9, based on the measurements presented in the supplementary information (Figure S3).
Comparison with instabilities during melt spinning
There are already different manufacturing instabilities described for common melt spinning including draw resonance and melt fracture which also lead to a fibre diameter oscillation. As in the case of melt spinning, molten polymers are pressed through spinnerets but in contrast to MEW, an uptake wheel draws the solidifying polymer fibres by mechanical stretching only. For melt spinning, the draw resonance is usually described as an instability occurring if a critical drawdown ratio (defined as fibre uptake speed divided by melt extrusion speed) is exceeded, ultimately leading to fibre breakage , , , . In this context, draw resonance was also described as partially disappearing at a second stable region or at high drawdown ratios, also depending on the spinning length (distance between spinnerets and uptake wheels for melt spinning) , , , , . Nevertheless, draw resonance cannot be readily compared to MEW due to the vastly different process conditions. MEW involves a different experimental set-up including a relatively small spinning length (collector distance) and the application of an accelerating voltage that both draws the jet and stabilises the fluid from jet break up. Most importantly, the very low mass flow rate required to establish a stable MEW process – typically 20–50 μL/h ,  and as low as 5 μL/h  is significantly lower compared to industrialised techniques such as melt spinning. Thus, the comparison of these two unstable melt operating regimes is speculative without further investigation.
Stable printing conditions
In order to print regularly structured scaffolds with MEW, it is important to avoid continuous pulsing or long beading (Figure 10). Due to changes in the CTS during fibre diameter oscillation, the scaffold quality decreases, especially at the turning loops. The jet and fibre drag is influenced by the homogeneity of the jet diameter and can lead to inaccurate fibre deposition. Figure 10A–B, G–H shows defective scaffold structures as a result of changes in printing direction during long beading conditions. Obviously, this instability needs to be prevented to enable stable printing conditions. Indeed, the feeding pressure and acceleration voltage effect on the deposition quality was shown, however, the influence of polymer characteristics, processing temperature, collector distance, spinneret size and environmental conditions should be investigated in order to optimise MEW over a broader range of conditions.
Fibre pulsing during MEW is a common phenomenon that occurs due to a force imbalance leading to mass flow oscillations and therefore to inhomogeneous fibre diameters. Since MEW has multiple instrumental parameters that affect fibre collection, balancing the parameters responsible for the jet stability can be challenging. Consistent diameters of the fibres are important for the fabrication of precise scaffolds, since jet homogeneity influences fibre placement. We demonstrate that pulsing can be observed by utilising the difference in the variable lag that follows the oscillation of jet thickness at the edge of simple collector movement paths. Long beading is a result of an extreme oscillation of mass flows that can be analysed by sorting and evaluating a sufficient number of measured fibre diameters. In order to avoid these undesired process instabilities, it is suitable to limit the mass flow through the spinneret by decreasing the feeding pressure or by stabilising the mass flow of the polymer jet by increasing the electrical field strength. Our presented approach to the operation of a MEW device should allow the user to identify pulsing and diminish this phenomenon to improve scaffold quality.
We would like to thank the European Research Council (ERC) (consolidator grant Design2Heal, contract # 617989) for their financial support and the Australian Technology Network - German Academic Exchange Service (ATN-DAAD) for their support of JNH. The authors would like to thank Dr. Claus Moseke, Ms. Judith Friedlein, and Mr. Joachim Liebscher for the technical assistance. TJ designed and assembled the MEW device. Further, we gratefully acknowledge financial support by the German Research Foundation (DFG) State Major Instrumentation Programme, for funding the Zeiss Crossbeam CB 340 scanning electron microscope (INST 105022/58-1 FUGG).
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About the article
Published Online: 2016-05-19
Published in Print: 2016-09-01
Conflict of interest: Authors state no conflict of interest.
Materials and methods
Informed consent: Informed consent has been obtained from all individuals included in this study.
Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the authors’ institutional review board or equivalent committee.
Citation Information: BioNanoMaterials, Volume 17, Issue 3-4, Pages 159–171, ISSN (Online) 2193-066X, ISSN (Print) 2193-0651, DOI: https://doi.org/10.1515/bnm-2015-0022.
©2016, Paul D. Dalton et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0