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

摘要

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.

摘要

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.

引言

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 flat^4,5 or a mandrel^6,7 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(ε-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 °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 °C and 82 °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).

方案

1. Material Preparation

1. Fill 2 g of PCL in a 3 mL plastic syringe with a funnel and insert a piston into the open end.
2. Place the syringe in a preheated oven at 65 °C for 8 h. Point the tip upwards to allow the air bubbles to aggregate close to the opening.
3. Push the piston with a thin object to release the trapped air within the molten material.
4. Let it cool down to room temperature, which is achieved when the polymer is not transparent anymore after 10 minutes.
5. Store the PCL pre-loaded syringe at room temperature in a dry and dark environment until it is used.

2. Hardware and Software Setup

1. 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.
2. 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.
3. 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.
4. 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.
5. Adjust the temperature regulators at the electrical box to 82 °C and 65 °C for the spinneret region and the syringe, respectively and power them on to melt the PCL.
6. Wait for at least 10 min until the polymer is molten and initiate the air pressure by setting the regulator to 1.8 bar.
7. 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).
8. Double-check manually that all ground cables are connected securely to the enclosure and the wall plug.
9. Start the software (e.g., MACH 3) on the computer and upload the prepared G-code.

3. Scaffold fabrication

1. 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.
2. 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).
3. 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.
4. Use the cursors on the keyboard to move the print head above the point where the G-codes will start.
5. Start the G-Code in the software on the computer.

4. Fiber Diameter Adjustment

1. Keep the working distance (12 mm) and the temperature regulators (82 °C and 65 °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.
2. Print fibers with small sized diameters (3-10 µm). 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.
3. Print fibers with medium sized diameters (10-20 µm). Adjust the air pressure level to 1.5 bar, set voltage to 11 kV and lower the collection speed to 1200 mm/min.
4. Print fibers with large diameters (20-30 µm). 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.

5. Jet Optimization

1. Illuminate the jet with a strong LED light from outside the enclosure for improved visibility.
2. 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.
3. 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.
4. 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.
5. 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.

6. Scaffold Collection

1. Open the door when the print is finished and use the cursor to move the collector plate towards the door for better accessibility.
2. Spray the scaffold with ethanol 70% (vol/vol) mix and wait 10 seconds until it visibly detaches from the collector.
3. Collect the finished scaffold by grabbing one edge with tweezers and lifting it out of the enclosure.

7. Troubleshooting

1. Decrease the applied voltage or open the door immediately if there is a spark between the spinneret visible or a cracking noise audible.
2. 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.
3. 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.
4. 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.

结果

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...

讨论

Integrating AM in order to find innovative solutions for the challenges in the medical field presents a new paradigm for the 21^st century. The so-called field of "Bio-fabrication" 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 ...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
塑料注射器	Nordson Australia Pty Ltd	7012072	EFD BARREL O 3mL Clear 50
医用级聚 （ε-己内酯） （mPCL）	Corbion Purac，荷兰	PURASORB PC12
23 GA 针	Nordson Australia Pty Ltd	7018302	#23GP .013 X .25 橙色 50 PC
柱塞	Nordson Australia Pty Ltd	7012166	活塞 O 3mL WH雨刮器 50
压力适配器	Nordson Australia Pty Ltd	7012059	适配器 ASM O 3mL BL 1.8M
铝收集器	Action Aluminium， Australia	SHP2	Sheet 5005 H34
亚克力玻璃	Mulford Plastics Pty Ltd	ACC6-13094
Mach 3 软件	Art Soft		在线购买
安全开关联锁	RS 组件Pty Ltd	12621330
高压发电机	EMCO 高压有限公司	DX250R
温度控制器	WATLOW	PM9R1FJ
X 和 Y 定位滑块	VELMEX Inc.	XN-10-0020-M011