Fabrication of 3D Polycaprolactone Macrostructures by 3D Electrospinning

Abstract

Building 3D electrospun macrostructures and monitoring the biological activities inside them are challenging. In this study, 3D fibrous polycaprolactone (PCL) macrostructures were successfully fabricated using in-house 3D electrospinning. The main factors supporting the 3D self-assembled nanofiber fabrication are the H₃PO₄ additives, flow rate, and initial distance. The effects of solution concentration, solvent, H₃PO₄ concentration, flow rate, initial distance, voltage, and nozzle speed on the 3D macrostructures were examined. The optimal conditions of 4 mL/h flow rate, 4 cm initial nozzle–collector distance, 14 kV voltage, and 1 mm/s nozzle speed provided a rapid buildup of cylinder macrostructures with 6 cm of diameter, reaching a final height of 16.18 ± 2.58 mm and a wall thickness of 3.98 ± 1.01 mm on one perimeter with uniform diameter across different sections (1.40 ± 1.10 μm average). Oxygen plasma treatment with 30–50 W for 5 min significantly improved the hydrophilicity of the PCL macrostructures, proving a suitable scaffold for in vitro cell cultures. Additionally, 3D images obtained by synchrotron radiation X-ray tomographic microscopy (SRXTM) presented cell penetration and cell growth within the scaffolds. This breakthrough in 3D electrospinning surpasses current scaffold fabrication limitations, opening new possibilities in various fields.

1. Introduction

Electrospinning is a simple and versatile technique for micro- and nanofiber fabrication using an electric force.¹ It employs an electric force to elongate the polymer solution, resulting in the formation of nanofibers.^2,3 The steps of nanofiber fabrication are illustrated in Figure 1a: (i) the solution droplet is charged and then transformed to the Taylor cone; (ii) the charged jet emerges from the Taylor cone and gets longer, which is called a straight jet; (iii) the jet is thinned in the electric field, and electrical bending instability appears and increases, called a whipping jet, whose length depends on the types and properties of materials and other applied parameters.⁴⁻⁷ Electrospinning allows nanofiber fabrication from various polymers and polymer-based materials, including metal, ceramic, carbon, and protein.^8,9 Electrospun fibers have been proposed to possess distinctive features, including a high surface-to-volume ratio, high porosity, interconnectivity, and biomimicry.⁹⁻¹² These features with morphology and alignment can also be easily adjusted by varying solution properties, electrospinning parameters, and modified collectors.^8,9 With the features mentioned, their potential has been approved in numerous applications, such as tissue engineering, drug delivery, wound healing, and energy storage. Even though electrospinning is a great way to produce nanofibers with great potential, the electrospun fiber can only be spared out in space as a flat, nonwoven, or two-dimensional (2D) mat. Instead of 2D mats, three-dimensional (3D) structures have presented more desirable results for some applications, such as biomedical applications,^9,13−15 sensors,¹⁶ and energy applications.⁸ In particular, the 2D mat presents low thickness and dense packs, resulting in too few areas for cell migration and infiltration.^9,13,14 The cells grew better on 3D electrospun scaffolds, especially in the long term of culture.¹⁵ In this regard, several approaches to 3D fibrous structures based on electrospinning have been proposed, for instance, multilayering electrospinning,¹⁷ postprocessing of the 2D mat,^18,19 mockup-assisted collectors,^20,21 modified collectors,²² and self-assembly.^8,14,23,24 Almost all approaches require several steps, additional complications, and time to achieve 3D fibrous structures, whereas self-assembly is a single step to obtain 3D structures. Self-assembly is mentioned to describe the formations of the 3D stack by continually accumulating fiber with a spatial configuration.^14,24 Herein, the self-assembly in a field of 3D fibrous constructs can be used to refer to the autonomous ability of the fiber to stand upright without any external support. For example, the quick fabrication of 3D nanofiber stacks and the controllable conversion between the 3D stack and 2D mat via conventional electrospinning was discovered.¹⁴ 15 wt % of polystyrene concentration was prepared in a mixture solvent of dimethylformamide (DMF) and tetrahydrofuran with a ratio of 1:1 by weight. The solution was fed at a rate of 0.5 mL/min under a voltage of 20 kV applied between a nozzle and an aluminum foil collector placed 15 cm apart. It was found that polystyrene fiber grew upward to form a 3D self-assembled stack as high as 100 mm in a short time. The mechanism behind the stack revealed that the fibers on the collector were still negatively charged because of electrostatic induction and polarization under a strong electric field, resulting in a new collector for the incoming fibers. The conversion between the 3D stack and 2D mat was achieved by inserting an insulating lucite plate on the collector to change from the 3D to the 2D mat and connecting the cathode of an electrostatic generator to the 2D mat to revert from the 2D to the 3D stack.¹⁴ The self-assembly mechanism has employed rapid solidification, electrostatic induction, and polarization.^3,9,14

Figure 1.

Comparative illustration of (a) ranges of fiber deposited in various techniques and (b) manufacturing concepts of these techniques including (i) conventional electrospinning, (ii) 3D printing, (iii) near-field electrospinning, (iv) melt electrowriting, and (v) 3D electrospinning.

The discovery of self-assembly has been developed by controlling the position of the depositing fiber following the model. Vong, Speirs, Klomkliang, Akinwumi, Nuansing, and Radacsi⁸ were successful in fabricating and controlling the shape of 3D polystyrene structures without the aid of any auxiliary template by using 3D electrospinning, which is a combination of 3D printing and electrospinning, to build up a 3D macrostructure and control its shape. 3D electrospinning relies on the fiber fabrication of electrospinning, including the stretching and whipping regions, to form the structures manufactured layer by layer, which is the base of 3D printing. Figure 1 shows schematic images of various techniques, including conventional electrospinning, 3D printing, near-field electrospinning, melt electrowriting, and 3D electrospinning. These techniques, except 3D printing, employ voltage to stretch the droplet of melted polymer or solution to form structures. The near-field electrospinning and melt electrowriting collected the fiber from the straight jet, which differs from the 3D electrospinning. The near-field electrospinning achieves controllable fiber deposition by reducing the distance to eliminate whipping.^6,25 Similarly, the whipping zone is absent in melt electrowriting because of the high viscosity and low charge density caused by using a polymer melt.²⁶ The near-field electrospinning and melt electrowriting have mainly been used in the buildup of micro- or milliscale structures with precise positioning, while 3D electrospinning has been used to build macrostructures at a scale of centimeters (Figure 1b).

3D electrospinning has been reported through the controllable fabrication of 3D polystyrene and polyvinylpyrrolidone structures; however, there is still room for improvement because there are many associated factors in the building-up process. In this work, a self-assembly approach for 3D fibrous macrostructures via 3D electrospinning, without the requirement of any support, has been studied by varying solution factors and electrospinning factors to observe the morphological characteristics and final shape of the macrostructures. Height or thickness is a criterion between 2D mats and 3D structures. Moreover, other parameters like convergence at half height (C_hh), wall thickness, and the width of deposition were defined to explain the effects of factors. Among the spinnable materials, polycaprolactone (PCL) was chosen to investigate the 3D buildup and its bioactivity assessment because it provides suitable biodegradable properties and is approved by the Food and Drug Administration for human medical applications.^9,20,27 A surface modification using plasma treatment is used to improve the low hydrophilicity of 3D PCL scaffolds.²⁸ The influence of the plasma treatment on the bioactivity is then revealed. Besides, the cell morphology and cell adhesion on the plasma-treated scaffolds are investigated by the synchrotron radiation X-ray tomographic microscopy (SRXTM) technique compared with scanning electron microscope (SEM) images.

2. Experimental Section

2.1. Materials

PCL (M_n = 80,000 kg mol^–1) and DMF (≥99.9% purity) were purchased from Sigma-Aldrich (United Kingdom). Orthophosphoric acid (H₃PO₄) (85% solution in H₂O) and dichloromethane (DCM) (greater than 99.5% purity) were both acquired from Loba Chemie, India. The chemicals were used without further purification.

Fibroblast (NIH3T3) cells used for in vitro cell assays were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S), 0.25% Trypsin-EDTA, and 10X phosphate-buffered saline (PBS) solution were purchased from Gibco (Waltham, MA, USA). Glutaraldehyde 25% in aqueous solution and osmium tetroxide 2% in aqueous solution (OsO₄) were obtained from Electron Microscopy Sciences and Thermo Fisher Scientific (USA), respectively.

2.2. Solution Preparation

PCL solutions were prepared by mixing PCL pellets with a blended solvent that contained DCM, DMF, and H₃PO₄ additives, then stirring the mixture for 24 h under ambient conditions. The solvent was prepared by various ratios of DCM:DMF that were 4:1, 6:1, and 8:1 and then added with H₃PO₄ additives by stirring for at least 10 min. The PCL was then added to the prepared solvent as follows: 10, 12, and 14 wt %. The PCL solutions were prepared with the same amount throughout all experiments, and all solutions were rested overnight before being used to spin fibrous structures via 3D electrospinning. The different conditions of solution preparation, including PCL concentration, solvent ratio, and H₃PO₄ additives, were used to investigate the 3D fibrous formation with a variety of flow rates, which were 2, 4, and 6 mL/h at 6 cm of the initial distance and under 16 kV of voltage as summarized in Table 1. Each electrospinning experiment was carried out for 30 min.

Table 1. Solution Preparations Investigated to Form 3D Fibrous Structures with Flow Rates of 2, 4, and 6 mL/h at an Initial Distance of 6 cm and Under an Applied Voltage of 16 kV via 3D Electrospinning.

parameters	unit	variable values
PCL concentration	wt %	10, 12, 14
solvent mixture (DCM:DMF)	by weight	4:1, 6:1, 8:1
H3PO4	by weight compared to PCL weight	0, 5, 10, 15, 20

2.3. 3D Electrospinning Apparatus and Process

A model and setup of the 3D electrospinning apparatus, which was designed and customized by a combination of electrospinning and 3D printing, are shown in Figure 2. It was modified, based on the Rostock-mini model of a delta-fused deposition modeling 3D printer, as follows: (1) the tower’s height was increased to 600 mm; (2) a Cu circular-flat plate with a diameter of 180 mm and a thickness of 1.6 mm was used to be a collector instead of the bed of 3D printer; (3) the linear bearings (attached to the carriages that connect to delta arms used to control the platform) were changed from parallel locomotion to a single one; and (4) the filament-extruder system was changed as a solution-feed system including a syringe pump (TE-331, Terumo, Terufusion), a syringe (20-mL syringe, NIPRO), a blunt nozzle (20Gx1 1/2″, NIPRO, cut to obtain a total length of 2 cm), a Teflon tube (inside diameter of 2 mm and an outside diameter of 4 mm), a connector between the syringe and the Teflon tube (a glass tube with an inside diameter of 4 mm, an outside diameter of 6 mm, and a length of 60 mm), and a nozzle holder (a printed model). White silicon tubes were used to cover the bottom parts of the strain steel towers to prevent electric sparks and fiber deposition on the steel.

Figure 2.

A prototype and experimental setup of 3D electrospinning, a combination of an electrospinning and a delta printer (Rostock mini), by modifying the tower, platform, and extrusion. These numbers refer to the following: 1 = syringe pump, 2 = syringe, 3 = connector, 4 = Teflon tube, 5 = nozzle holder, 6 = platform, 7 = high-voltage source, 8 = collector, 9 = carriage, 10 = tower end, 11 = delta arms, 12 = idler end and a limit switch, 13 = thermometer and hygrometer, 14 = ventilation fan, and 15 = heater.

The 3D electrospinning apparatus was driven on a Ramp1.4 board by a modified Repetier firmware. The apparatus was within the chamber (85 × 60 × 70 cm³), which comprised a closable/openable door (a customized acrylic sheet), a hot-air heater, a ventilation machine, and a thermo-hygrometer. A high-voltage (HV) DC power supply (CY-DW-P503–1ACDF, Zhengzhou CY Scientific Instrument Co. Ltd., China; 50 kV DC output voltage and 1 mA output current) was separately equipped. It has been used to generate a potential difference between the nozzle and the collector. This setup allows the fabrication of objects 180 mm in diameter and 300 mm in height. All parameters were controlled through the LCD screen.

Before doing experiments, a 3D model is able to be designed with various software programs like Tinkercad, Blender, and Autodesk Fusion360 and then sliced through the Repetier slicer software. The initial distance and nozzle speed were determined by adjusting the Z-offset and travel speed in the software. The collector was wrapped in aluminum foil. The polymer solution was loaded into the syringe connected with a connector, a Teflon tube, and a blunt nozzle. At the start of the experiments, the positive charge and ground of the voltage source were connected to the nozzle and collector, respectively.

To investigate the buildup of the 3D fibrous macrostructures, a hollow cylinder with a diameter of 60 mm was printed as a model. The model file is sliced with a fixed layer height of 0.4 mm and one perimeter to gain a motion pattern on a gcode file. The 3D electrospun structures were tested by varying flow rates, initial distances, voltages, and nozzle speeds, as summarized in Table 2. The solution flow rate and applied voltage were separately controlled through the syringe pump and the high-voltage source. All experiments were performed between 30 and 35 °C, and the relative humidity was imposed between 40% and 50%. Each experiment ran for 30 min.

Table 2. Electrospinning Parameters Used to Form 3D-Fibrous Macrostructures Using PCL Solution via 3D Electrospinning.

parameters	unit	variable values
flow rate	mL/h	2, 3, 4, 5, 6
initial distance	cm	2, 3, 4, 5, 6
high voltage power supply	kV	12, 13, 14, 15, 16
nozzle speed	mm/s	1, 2, 3, 4, 5

2.4. Structural and Morphology Characterization

A digital camera was used to record the buildup of the 3D self-assembly and monitor the development of the final product. The SEM (JEOL JSM-6010LV, Japan) was used to observe the fiber morphology. Prior to observation, the samples were coated with a layer of gold for 2–3 min, resulting in a thickness of around 30 nm, using the gold sputter coater (Neo Coater/MP-19020NCTR). Each SEM image was divided into 3 × 3 regions, and the diameters of five fibers in each area were measured using ImageJ software (National Institutes of Health, MD, USA). Hence, the fiber diameter of each sample was averaged by assessing 135 fibers across three distinct areas. In this work, a criterion is used to differentiate between a 2D mat and a 3D fibrous structure, wherein a height exceeding 10 mm is designated as indicative of a 3D structure. For the diameter measurement of 3D fibrous structures, a sample was observed at three distinct locations: the upper, middle, and lower parts. The average diameter of each part was measured from 135 fibers using the mentioned method. Therefore, to find the average diameters of the whole 3D fibrous structure, measurements were taken from 405 separate fibers collected from nine different areas of the upper, middle, and lower parts. The diameter distribution of a 3D fibrous structure was assessed by calculating the average diameters of each part, employing the p-value statistical method.

2.5. Surface Modification by Plasma Treatment

The obtained 3D PCL-electrospun structures were modified by plasma treatment. The treatment was performed using a plasma generator (Surface Technology HF Generators 200 W, Diener Electronics, Germany) at the Synchrotron Light Research Institute (a public organization) in Thailand. O₂ gas was chosen to generate the plasma. The chamber was filled with gas prior to the treatment. The power intensities, including 10, 30, 50, and 70 W, were applied for 5 min to investigate the bioactivity of the surface modification. The pressure of the plasma chamber was kept at 0.2 mbar under all conditions by controlling the gas flow.

2.6. Biological Assays on the Surface Modification

The untreated and plasma-treated scaffolds were prepared as 5 × 5 mm² scaffolds for biological assays. A glass slide and untreated and plasma-treated scaffolds were placed in a 12-well plate (Costar, Corning, USA). The samples were sterilized under ultraviolet light for 30 min. Then, all were washed with cell culture media, containing DMEM, 10% FBS, and 1% P/S, for 1 h and further soaked in the media overnight to tune the environment onto 3D scaffolds. 2 ×10⁵ fibroblast NIH3T3 cells were added to the the glass slide, which was used as a control, and all scaffolds. The media were then added until each well contain 1 mL and were not changed during the 3-day culture at 37 °C with 5% humidified CO₂. To assess the cell adhesion and morphology, after 3 days of cell culture, the control and scaffolds were washed twice with 1 mL of 1X PBS and then fixed with 2.5% glutaraldehyde (1 mL/well) in PBS for 2 h at room temperature. All samples were further washed twice with 1X PBS for 10 min before being dehydrated through a series of ethanol solutions, including 30%, 50%, 75%, and 99%, for 5 min. Prior to SEM examination, the dehydrated samples were stored overnight and then coated with gold, approximately 10 nm in thickness, using sputter coating equipment (Sputter Coater, Leica/EM ACE600). SEM (FESEM-FIB/EDS-Carl Zeiss/AURIGA) was used to observe the cell adhesion and morphology. The optimized plasma condition was used to prepare scaffolds for further observation of cell morphology and adhesion with the SRXTM technique.

2.7. Biological Assays Using Synchrotron-Based X-ray Tomography Microscopy (SRXTM)

The 3D electrospun structure with a plasma treatment of 50 W was prepared as 5 × 5 mm² to use as scaffolds in the biological assays. The glass slide (a cell control) and plasma-treated scaffolds were placed in 12-well plates and sterilized as per the mentioned protocol. 2 ×10⁵ fibroblast NIH3T3 cells were seeded on the glass slide and plasma-treated scaffolds. A plasma-treated scaffold was also put in the plates without seeded cells (used as a fiber control). The media were added into each well as 1 mL. After 1 and 3 days of culture, the controls and scaffolds were washed with the PBS solution and fixed with glutaraldehyde, as per the protocol mentioned. After the fixation, the samples were stained with 1% osmium oxide solution for 4 h before being washed with the PBS solution and dehydration. Each sample was cut to prepare a suitable size for a 3.0 mm-diameter Kapton tube, and the tube was glued onto a pin holder.

SRXTM was used to observe cell distribution inside fibrous scaffolds that can imply the visualization of cell migration and measure the normalization of cell volume between the samples cultured for 1 and 3 days. The experiment was executed at an SRXTM beamline (beamline 1.2 W: X-ray imaging and tomographic microscopy) at the Synchrotron Light Research Institute in Thailand. The synchrotron radiation was originated from a 2.2 T multipole wiggler at the 1.2 GeV Siam Photon Source. By using a filtered polychromatic X-ray beam, the experiments were executed at a mean energy of 14 keV with a 32-m distance from the source to sample. All tomographic scans with the pco edge 5.5 detector at 5× total magnification were performed. The field of view was 3.70 × 3.12 mm with a pixel size of 1.44 μm. Each sample prepared for the holder was mounted on a rotary stage. Then, a total of 2001 X-ray radiographs were obtained from 0° to 180° with an angular increment of 0.1°. The obtained radiographs were normalized by flat-field correction with open-beam and dark images and then reconstructed using Octopus Reconstruction software (TESCAN, Gent, Belgium).²⁹ After obtaining the reconstruction images, the 3D data set was converted from 16-bit TIFF images into 8-bit images using ImageJ software. The image processing was computed using Drishti software.³⁰ The Kapton tube seen around the scaffold material was cropped out to reduce the size of the data. The difference between the intensity of the stained cell and the polymer was used to identify the cells and scaffolds. Both the original PCL fibers and the fiber control were investigated using SRXTM with the same protocol. In addition, the measurement function of volume was used to measure the volume of the fibers and cells. Since the number of fibers was different on electrospun scaffolds, the normalized volume of cells was calculated by comparing them with the volume of fiber.

3. Results and Discussion

3.1. The Buildup of 3D Macrostructures by Using 3D Electrospinning

In this work, the optimal conditions for building up the 3D electrospun macrostructure via 3D electrospinning have been studied. The specific criterion required to distinguish between a 2D mat and a 3D structure is thickness or height. Generally, the electrospun fibers obtained from conventional electrospinning range from nano-, micro-, to millimeter scale. Therefore, a sample greater than 10 mm is considered to be an indication of 3D structures. Figure 3a illustrates the 3D buildup at discrete intervals of 5 min, and more different time intervals are demonstrated Video S1. The buildup of the 3D electrospun fiber self-assembly spontaneously occurred within 1 min after starting the process, as shown in Figure 3a. Initially, a 2D mat was formed at the location that only corresponded to the nozzle for 2–3 s. After that, the fibers started to rise between the nozzle and the collector. The raised-up fibers were able to move following the nozzle movement to build up and control a 3D structure. There was some delay between the deposited fibers and the movement of the nozzle. This observation resembles the jet lag found in the melt electrowriting process (Figure 3a) at 1, 5, 10, and 15 min).²⁵ As the structures got higher, the fibers fell to the edges of the structure (Figure 3a). It was slightly different at the initial stage from the previous report, where 3D polystyrene formation happened after first depositing an initial layer, the flat layer of 2D fibers.⁸ It might be attributed to the different conductivities of solutions. The previous study reported that a 3D stack made from a polystyrene solution was generated on a fiber mat, while one made from the polystyrene solution with an additive was more likely to get fast growing without the mat.¹⁴ However, the flat layer at the bottom part of the 3D fibrous structures in this work remains. It was generated during the 3D formation because of whipping instability, resulting in the fibers depositing at the edges of structures and surrounding areas, as obviously shown in Figure 3a at 20 min. It caused the convergent shape at the bottom part of the structures. The flat layer at the bottom resembles a raft or a brim in 3D printing (as shown in the adhesion types of 3D printing in Figure 3b). The raft and brim are types of adhesions that are adjustable to avoid warping. Nevertheless, it cannot be adjusted via 3D electrospinning because of the inherent whipping jet.

Figure 3.

(a) The buildup of 3D fibrous macrostructures at a flow rate of 6 mL/h, an initial distance of 6 cm, a voltage of 16 kV, and a nozzle speed of 1 mm/s by an in-house 3D electrospinning setup and (b) schematic images of the perimeters and adhesion types in 3D printing, the jet lag in melt electrowriting, and the 3D electrospinning.

The deposited fibers, being negatively charged, act as the new collectors for the incoming fibers because of polarization and electrostatic induction according to the self-assembly mechanism.^8,9,14,31 As seen in Figure 3a, the built-up fibers, attracted by the positively charged nozzle, were dragged along the pathway of the nozzle. Some fibers were then moved downward to the previously deposited area and stacked to form a 3D structure. During the electrospinning process, some collapse and overhang occurred because of its weight at the accumulation position and whipping instability.²⁴

This is how the 3D fibrous macrostructures form via 3D electrospinning. It is unattainable to achieve a structure identical to the model due to the inherent whipping instability in the electrospinning process. To understand the associated factors, varying solution preparations and electrospinning parameters are investigated, and the final products are assessed as shown in Figure 4. A thickness was measured as a height to identify a 2D mat and a 3D structure. The width of deposition is measured from a length from one side at the bottom part of 3D structures to another. The wall thickness is measured by the difference between the inside diameter and the outside diameter (D_out). The wall thickness is considered in the case of the perimeter to be 1. The C_hh is also measured to assess the deformation in the model.

Figure 4.

(a) Diagrams and (b) examples of parameters used to illustrate 3D electrospun structures in this work. The scale bar is 10 mm.

3.2. Solution Parameters

There are several factors that affect the buildup of 3D macrostructures via 3D electrospinning. The PCL solutions with various conditions of solution preparation were investigated to explore the influence of concentrations, solvent ratios, and additives on the buildup of the 3D electrospun structures. The relations between various solution parameters and flow rates were investigated. 10, 12, and 14 wt % concentrations of PCL solutions prepared in a solvent containing a DCM:DMF ratio of 4:1, 6:1, and 8:1 and an addition of 5–20% H₃PO₄ were used to survey the optimal solution with the flow rates of 2, 4, and 6 mL/h, at an initial distance of 6 cm, a voltage of 16 kV, and 1 mm/s nozzle speed under ambient conditions for 30 min. The side view of all final fibrous constructs obtained is shown in Figure S1.

3.2.1. Relation between Concentration and Flow Rate

In the ranges of concentration, the 12 wt % concentration was preferred for the buildup of 3D PCL-electrospun macrostructures that resembled the model. It presented the highest structures, reaching 32.30 ± 3.90 mm. No 3D electrospun construct was found under the 10 wt % concentration. All performances of 10 wt % had unstable electrospinning jets and solution drying, leading to hampering the self-assembly and electrospinning processes. At low concentrations, the charged jets lose their intermolecular attractions and are thus discrete into droplets from the Taylor cone due to low viscosity and high surface tension.^7,11 When the concentration was increased to 12 wt %, it provided stable electrospinning and no drying. Increasing the concentration will lead to an increase in chain entanglement among the polymer chains, resulting in stable 3D structures.^2,7 However, increasing the concentration beyond the threshold causes the droplet to dry at the tip as well, resulting in an interruption of the electrospinning process and a decrease in height, because the increase in concentration leads to an increase in viscosity.^32,33 The high concentration also has the possibility of a shape-shifting effect, as seen in the top view of the constructs in Figure S2, because of the weight of the deposited fibers.

Generally, the increase in concentration increases the diameter of the electrospun fibers,^2,3,8 but that was not the case in this study. The SEM images of all electrospun fibers are presented in Figure S3. An increase in concentration from 10 to 12 wt % increased diameters; however, the average diameter of 14 wt % decreased, as seen in Figure S4. It was attributed to the change in electrospinning distance. If there are conditions that cannot perform the 3D formation, it means that the distance will increase gradually. In other words, the lower height of the 3D structure means a longer distance of electrospinning, which might affect the voltage potential. Even though no 3D formations were found at 10 wt % concentration, the dripping and drying found were able to explain the bigger fiber.

3.2.2. Relation between Solvent Ratio and Flow Rate

The solvent is another important factor in the 3D self-assembly because the formation employs rapid solidification alongside electrostatic induction. A high evaporation rate of volatile solvent is desired; however, a high evaporation can cause the solution to dry at the nozzle tip, resulting in clogging.²

The only solutions prepared using the 6:1 solvent ratio performed stable electrospinning processes. An increase in the DCM ratio resulted in a wider wall thickness, as seen in the top view of the electrospun structures in Figure S5. In near-field electrospinning, a high amount of DCM in the solvent performed the random deposition of PCL fibers due to the whipping effect, and a decreasing amount of DCM would offer straight fibers.³⁴ Because the high evaporation rate of DCM increased surface tension on a droplet, a large amount of charge was required to overcome the surface tension, leading to repulsive forces and the whipping effect.³⁴ Similarly, an increase in the DCM in the solvent ratio increased the deposition area at a flow rate of 2 mL/h. However, it changed when the structure was moved upward because the fiber on the 3D structure was moved from the flat collector to the edge and top of the structures.

The morphologies of the 2D mats and 3D structures are shown in Figure S6. It was found that the average diameter (Figure S7) of the structures was related to the structure height. The higher structures had a larger diameter because the top part of the structures was closer to the nozzle. It corresponds to the relation of concentration.

3.2.3. Relation between Additives and Flow Rate

Among these solution parameters, the addition of the H₃PO₄ additive has played a crucial role in the formation of 3D PCL-fibrous macrostructures, as seen in Figure 5. It appeared that the lack of additives prevented the fiber from being formed in the 3D structures. It is in accordance with the results reported in previous studies.^9,35 The pure PCL solution can only produce a 2D fibrous membrane, and the incorporation of lactic acid into the PCL solution aimed to form 3D sponge-like structures.⁹ The mechanism behind the self-assembled formations of electrospun fibers relates to polarization and electrostatic induction of the deposited fibers.^8,14,31 The addition of H₃PO₄ to the PCL solutions increased the conductivity of the solutions, as shown in Figure S8. The conductivity was measured using a conductivity benchtop meter (SevenCompact Duo S213 pH/conductivity, Mettler Toledo). The increase in conductivity increases the surface charge, enabling the effort of repulsive force, electrostatic induction, and polarization of the fibers being deposited, ultimately resulting in self-assembly to form 3D structures.^2,36 However, the relationship between 3D electrospinning and solution conductivity is not directly correlated, and the influence of other parameters cannot be disregarded.²³ Moreover, the number of additives directly affects the existing hollow shape of 3D electrospun structures under the constant parameters of the flow rate, initial distance, voltage, and nozzle speed, as shown in Figure S9. The increase of additives beyond a critical point led to the formation of fluffy structures and a reduction in the ability to control the shape because the repulsive forces of fibers increased, leading to a wider area of fiber deposition.

Figure 5.

Comparative side view images of PCL-fibrous constructs using 12 wt % solutions without and with the addition of 10% H₃PO₄ additives. The constructs were formed under the conditions of 2, 4, and 6 mL/h flow rate, 6 cm initial distance, and 16 kV voltage. The scale bar is 15 mm.

The SEM images shown in Figure S10 demonstrate an increase in the pore size, corresponding to the increasing proportion of H₃PO₄. Similarly, a previous study reported that the pore size of 3D fibrous scaffolds varies according to lactic acid contents.⁹ Furthermore, higher concentrations of additives induced a wider area of deposition and had an increasing trend in diameter, as shown in Figure S11. The excessive additives, including H₃PO₄ contents of 15 and 20%, obviously resulted in a lot of beads.

3.3. Electrospinning Process Parameters

The PCL solution with the 12 wt % concentration in 6:1 by weight DCM:DMF was used to investigate the electrospinning factors, including the flow rate of solution, the initial distance between a nozzle and a collector, the HV applied between the nozzle and the collector, and the nozzle speed, on the buildup of 3D PCL-electrospun macrostructures.

3.3.1. Flow Rate

A minimum flow rate is necessary for sufficient deposition of the fibers to perform the 3D buildup.³ A balance between the amount of the extruded solution and the replacement of the new solution during electrospinning is needed. The flow rate in a range of 2, 3, 4, 5, and 6 mL/h was used to study the 3D buildup under an initial distance of 6 cm, a voltage of 16 kV, and a nozzle speed of 1 mm/s.

As shown in Figure 6a,b, the 3D buildup occurred when using the flow rate of 4 mL/h, and the height of structures is likely to increase following an increase in the flow rate. The increase in flow rate is related to a higher amount of polymer solution extruded from the tip of the nozzle. As seen in Figure S12, an increasing amount of the raised-up fibers deposited onto the collector following an increase in flow rate was observed. However, the appearance of the depositing fiber changed from the vertically raised-up fibers to the horizontally widespread fibers when the 3D structures were unable to grow up in response to the rising distance between the nozzle and collector, as shown in Figure S12b,d. This resulted in losing control of the position of the depositing fibers and expanding the area of fiber deposition, finally achieving a 2D mat. The flow rate of 4 mL/h demonstrated sufficient depositing fibers to enable dragged fibers to follow the model throughout the 3D buildup. At the flow rate of 5 mL/h, the solution was drying and clogging occurred at the nozzle tip, resulting in discrete 3D buildup. Although normal self-assembly formations were performed after the dripping, the height of the obtained structures was decreased. At a flow rate of 6 mL/h, the raised-up fiber was drawn following the model at the initial stage. When the structure was higher, the raised fiber was shorter and the incoming fiber was deposited at the edge of the structure. This was attributed to the increasing amount of incoming fiber, which resulted in faster buildup than at 4 mL/h. Interestingly, a faster flow rate at the flow rates of 5 and 6 mL/h induced some shape-shifting, as seen in Figure 6a. A sufficient amount of incoming fiber is necessary to obtain charge induction, polarization effect, and repulsive force between fibers to build up; however, the excessive amount at the high flow rate decreases printing quality.^3,8 This work uses the flow rate of 4 mL/h as the optimal condition for the 3D buildup of a 12 wt % PCL solution.

Figure 6.

(a) Top and (b) side views of comparative 3D PCL-electrospun constructs by varying the values of a flow rate. The flow rate of solution, 12 wt % PCL solution in 6:1 DCM:DMF + 10% H₃PO₄, was increased from 2 to 6 mL/h, with each increment being 1 mL/h. The 3D PCL formations were performed at an initial distance of 6 cm, a voltage of 16 kV, and a nozzle speed of 1 mm/s for 30 min. (c) SEM images of the upper part of the electrospun mat and the upper, middle, and lower parts of the 3D structures. The scale bars indicated in (b) and (c) are, respectively, 10 mm and 50 μm.

As mentioned before, an increase in flow rate enables the rapid buildup of 3D macrostructures and is likely to result in an increase in their height. However, above the optimized flow rate, shape-shifting, structure collapse, and solution clogging were observed. The height of the structure starts to decrease at a flow rate of 5 mL/h due to the discontinued accumulation resulting from drying of the solution. The increase in flow rate resulted in a wider deposition area in the range of 2–3 mL/h, in which a 2D mat was obtained. It is in agreement with the previous findings.⁸ It was attributed to the inherent effect of parameter changes over time. Since the coming fibers at 2–3 mL/h were too small to build up, as time went on, the distance of electrospinning increased, leading to a wider area of deposition. The width of the deposition at 2 mL/h was 80.88 ± 1.33 mm and increased to 93.55 ± 7.10 mm when the flow rate was increased to 3 mL/h. Nevertheless, the width of deposition decreased as the structures were higher, as indicated in Table 3. The deposition width of the first layer was caused by the deposition of the electrospun mat in the initial stage. It was attributed to the continuous positioning of depositing fiber on the top and at the edge of structures during electrospinning. As the structure rises, some deposited fibers will deposit onto the edge of the structure instead of the collector. This results in increased C_hh and decreased D_out when height is higher. The wall thickness measured at the top of structures was thinner with an increase in flow rate of 8.28 ± 0.61, 6.05 ± 1.10, and 2.69 ± 0.57 mm for the applying flow rates at 4, 5, and 6 mL/h. An increase in flow rate induces a longer straight jet,³⁷ and an increased structure’s height enables balancing the electrospinning distance, resulting in no increase in the length of the whipping jet.

Table 3. Summary of the Overall Average Diameter of Fiber, the Height, C_hh, the Width of Deposition, D_out, and Wall Thickness of 3D Structures Obtained from Various Flow Rates, Including 2, 3, 4, 5, and 6 mL/h^a.

experimental conditions (kV-cm-mL/h-mm/s)	fiber diameter (mm)	height (mm)	Chh(mm)	width of deposition (mm)	Dout(mm)	wall thickness (mm)
16–6–2–1	2.44 ± 1.91	7.30 ± 0.12	3.88 ± 0.44	80.88 ± 1.33	61.68 ± 2.58	11.55 ± 3.10
16–6–3–1	1.83 ± 1.27	4.83 ± 1.43	-	93.55 ± 7.10	-	-
16–6–4–1	4.10 ± 3.16	23.50 ± 0.96	12.69 ± 1.09	81.10 ± 4.69	52.70 ± 1.56	8.28 ± 0.61
16–6–5–1	4.71 ± 3.97	15.30 ± 1.50	10.61 ± 0.23	81.20 ± 2.74	55.40 ± 2.13	6.05 ± 1.10
16–6–6–1	4.11 ± 3.31	32.25 ± 3.86	20.09 ± 2.78	71.88 ± 3.82	41.72 ± 0.88	2.69 ± 0.57

^aThe experimental conditions are in the following order: voltage, initial distance, flow rate, and nozzle speed.

The morphologies of varying flow rates used in the 3D formation via 3D electrospinning are shown in Figure 6c. The morphological characteristics of electrospun mat spun at flow rates of 2 and 3 mL/h were similar. Thinner fibers were intertwined with larger fibers. The average diameter at 2 mL/h was 2.40 ± 1.90 μm and decreased to 1.80 ± 1.30 μm when applying 3 mL/h. The small decrease in diameter at 3 mL/h might correspond to the slight decrease in the structure’s height, resulting in a little increase in the distance between the nozzle and the collector. A decrease in volumetric flow rate resulted to an increase in volume charge density and faster travel of the charged jet toward the collector, leading to thicker fiber.³⁸ As seen in Figure S13a,b, 3D macrostructures provided a wider range of diameter. The lower part of the 3D structures at flow rates of 4, 5, and 6 mL/h had the lowest average diameter. As a result, the middle and/or top parts that were closer to the nozzle had a larger diameter. The average diameter of the upper part at 4 mL/h was significantly different from those of the middle and lower parts. When the flow rate increased to 6 mL/h, the average diameter of the lower part was significantly different from those of the other parts. This was consistent with what happened during the 3D formation: the high flow rate resulted in faster buildup. The average diameter did not exhibit a rise with the increase in solution flow rate due to the wide range of diameter.

3.3.2. Initial Distance

An initial nozzle–collector distance is one of the necessary parameters in 3D electrospun buildup since it relates to the determination of buildup height.⁸ The electrospinning distance changes over time, following the nozzle movement. The 12 wt % PCL solution prepared in 6:1 DCM:DMF solvent was used to investigate a varying initial distance of 2 to 6 cm under a flow rate of 4 mL/h, a voltage of 16 kV, and a nozzle speed of 1 mm/s. Figure 7a,b shows the evolution of the 3D buildup by varying the initial distance.

Figure 7.

(a) Top and (b) side views of comparative 3D PCL-electrospun constructs by varying the values of an initial distance. The initial distance applied to run the formation procedure of a 12 wt % PCL solution in 6:1 DCM:DMF + 10% H₃PO₄ was increased from 2 to 6 cm and subsequently raised in increments of 1 cm. The 3D PCL structures were formed at a flow rate of 4 mL/h, a voltage of 16 kV, and a nozzle speed of 1 mm/s for 30 min. (c) SEM images of the upper part of the electrospun mat and the upper, middle, and lower parts of the 3D structures. The scale bars indicated in (a) and (c) are, respectively, 10 mm and 50 μm.

At initial distances of 2 and 3 cm, a 2D mat was obtained. The short nozzle–collector distance cannot allow for the 3D buildup, which was also found in a previous study.⁸ This can be attributed to the influence of a strong voltage and short distance. As seen in Figure S14a–d, the deposition of raised-up fibers was scattered in various positions. At the initial stage, the deposition pathway of spun fiber remained a hollow cylinder; however, it finally disappeared because of the widespread deposition, resulting in the circle mat (Figure 7b). There were also some drips and drying at the tip of the nozzle due to the short distance. It showed the initial distance at 2 and 3 cm is too short to form 3D fibrous macrostructures. The 3D buildup started at the initial distance of 4 cm. The raised-up fiber, which enabled moving along the nozzle, was observed at the initial stage. It is important to note that there were raised-up fibers in two directions on previously fiber-deposited structures, as seen in Figure S14e,f. This effect was attributed to the multijet ejection of electrospinning. The multijet regime was found when decreasing the distance at a constant voltage, and a decreasing distance was likely to increase the number of multijet splits.³⁷ Likewise, the multijet ejection was found at the initial stage under the initial distance of 5 cm and disappeared when the nozzle moved upward. The unbalance of upward height between nozzles and structures resulted in widespread deposition and finally obtaining mats for the initial distance of 5 cm. Nevertheless, the multijet ejection was not noticeable at the initial distance of 6 cm. The built-up fiber containing many electrospun fibers enables nozzle movement in one position instead of wide deposition.

The increase in the initial distance allows for higher structures; however, it might risk being inconsistent and unstable because of the longer length of the whipping jet. The increase in initial distance increased the deposition areas, similar to the previously reported results.⁸ It was attributed to the natural consequences of the electrospinning process. Separately considering the 2D mat and 3D structures, the widths of deposition of the initial distance of 2, 3, and 5 cm were 85.76 ± 3.59, 91.37 ± 4.72, and 93.30 ± 2.53 mm. Similarly, the width of deposition increased from 78.95 ± 2.28 to 81.10 ± 4.69 mm when the initial distance was increased from 4 to 6 cm. Likely, the increase in initial distance allowed for a larger wall thickness, which was increased from 4.50 ± 2.26 to 8.28 ± 0.61 mm when increasing the distance from 4 to 6 cm. It is attributed to the longer jet in the whipping zone. The C_hh increased with the increase of the initial distance, and the D_out was wider with the increase of the initial distance, as concluded in Table 4.

Table 4. Summary of the Overall Average Diameter of Fiber, Height, C_hh, Width of Deposition, D_out, and Wall Thickness of 3D Structures Obtained from Various Initial Distances, Including 2, 3, 4, 5, and 6 cm^a.

experimental conditions (kV-cm-mL/h-mm/s)	fiber diameter (mm)	height (mm)	Chh(mm)	width of deposition (mm)	Dout(mm)	wall thickness (mm)
16–2–4–1	2.04 ± 1.76	0.20 ± 0.08	-	85.76 ± 3.59	-	-
16–3–4–1	2.98 ± 1.76	0.15 ± 0.06	-	91.37 ± 4.72	-	-
16–4–4–1	2.17 ± 1.44	13.90 ± 2.07	7.74 ± 3.19	78.95 ± 2.28	48.03 ± 2.66	4.50 ± 2.26
16–5–4–1	2.65 ± 1.92	8.50 ± 2.39	-	93.30 ± 2.53	-	-
16–6–4–1	4.10 ± 3.16	23.50 ± 0.96	12.69 ± 1.09	81.10 ± 4.69	52.70 ± 1.56	8.28 ± 0.61

^aThe experimental conditions are in the following order: voltage, initial distance, flow rate, and nozzle speed.

The morphology of the electrospun mats is shown in Figure 7c. The dense pack of fiber and some beaded fibers was found in the electrospun mat fabricated at 2 and 3 cm. As seen in Figure S15a, there was a significant difference between the lower and middle parts under the initial distance of 4 cm; however, it was changed to be the upper part, which was significantly different from others. It was probably because the short initial distance caused more deposited fiber and limited the height of the raised-up fiber, resulting in dense-pack fiber on the lower parts of the structures under the initial distance of 4 cm. When the initial distance was higher, it allowed for a faster buildup. However, if the distance between the upward-moving nozzle and the rising height of structures were not balanced, it would result in a significantly different size of the upper part. For the 2D mat, although there was an increase in diameter between 2 and 3 cm, there was a slight decrease when the initial distance increased to 5 cm. The large fiber in the range of 2 to 3 cm might relate to the solution dripping. The overall of the average diameter of 3D structures shows that the increase in initial distance is likely to increase the average diameter. It was attributed to the upward height of structures over time.

3.3.3. Voltage

The study of various voltages applied to build up the 3D PCL-electrospun structures was executed by applying a flow rate of 4 mL/h, an initial distance of 4 cm, and a nozzle speed of 1 mm/s for 30 min. The influence of voltage on the 3D formations is shown in Figures 8 and S16. At the applied voltage of 12 kV, the 2D mat with a thickness of 1.60 ± 0.36 μm was obtained with a controllable deposition shape, even though some raised-up fibers occurred and enabled the deposition following the nozzle locomotion. Due to the low voltage, some accumulation of droplet drying, which caused the prolonged droplet at the tip and the dripping on the collector, was observed.^3,8 The 3D buildup started at an applied voltage of 13 kV. As seen in Figure 8a,b, the structure rose to 15.95 ± 1.53 mm at 13 kV and reached its highest value at 14 kV. When the voltage was increased to 15 and 16 kV, the height of structures decreased. It might cause an increase in the jet velocity when applying a stronger voltage. An increase in applied voltage will result in a decrease in the size of the Taylor cone and an increase in the jet velocity.^2,3 The multijet ejection was observed when increasing the applying voltage to 15 and 16 kV, as seen in Figure S16. An increase in voltage at a constant distance induces multijet ejection since increasing voltage beyond the threshold results in the disappearance of a protruding droplet and electric field concentration on the tube edge, resulting in more than one jet.^37,39

Figure 8.

(a) Top and (b) side views of comparative 3D PCL-electrospun constructs by varying the values of a voltage. The voltage applied to run the formation procedure of a 12 wt % PCL solution in 6:1 DCM:DMF + 10% H₃PO₄ was increased from 12 to 16 kV, subsequently raised in increments of 1 kV. The 3D PCL structures were formed at a flow rate of 4 mL/h, an initial distance of 4 cm, and a nozzle speed of 1 mm/s for 30 min. (c) SEM images of the upper part of the electrospun mat and the upper, middle, and lower parts of 3D structures. The scale bars indicated in (b) and (c) are 10 mm and 50 μm, respectively.

The potential difference applied between the nozzle and collector relates to the force used to stretch the droplet. An increase in voltage increases the velocity of the jet onto the collector. An increase in applied voltage up to a certain point allows for a higher structure, but a voltage greater than that point leads to a decrease in the height of the structures, as concluded in Table 5. Likewise, C_hh corresponds to the height. For the width of deposition, D_out, and wall thickness, these decrease with an increase in voltage. It was attributed to the shorter length of the whipping jet. An increase in voltage results in an increase in the length of the straight jet and a decrease in the width of the whipping zone.³⁸ This caused a trend to decrease the wall thickness, which was 14.42 ± 1.36, 7.70 ± 1.75, and 3.98 ± 1.01 mm for 12, 13, and 14 kV; however, the increase in wall thickness at 15 and 16 kV was 8.63 ± 4.02 and 4.50 ± 2.26 mm, which might be attributed to the multijet ejections observed. Likewise, the D_out decreased with an increase in voltage; however, an increase in D_out was observed in the range of multijet ejection observed.

Table 5. Summary of the Overall Average Diameter of the Fiber, Height, C_hh, Width of Deposition, D_out, and Wall Thickness of 3D Structures Obtained from Various Voltages, Including 12, 13, 14, 15, and 16 kV^a.

experimental conditions (kV-cm-mL/h-mm/s)	fiber diameter (mm)	height (mm)	Chh (mm )	width of deposition (mm)	Dout (mm)	wall thickness (mm)
12–4–4–1	2.83 ± 2.33	1.60 ± 0.36	-	84.41 ± 11.46	67.59 ± 3.05	6.67 ± 1.06
13–4–4–1	3.09 ± 1.89	15.95 ± 1.53	11.95 ± 0.39	82.60 ± 2.27	46.55 ± 2.04	7.70 ± 1.75
14–4–4–1	1.45 ± 1.10	16.18 ± 2.58	15.49 ± 4.60	72.93 ± 2.70	43.85 ± 1.58	3.98 ± 1.01
15–4–4–1	2.74 ± 1.94	15.75 ± 2.64	9.94 ± 5.40	82.26 ± 4.50	52.98 ± 7.80	8.63 ± 4.02
16–4–4–1	2.17 ± 1.44	13.90 ± 2.07	7.74 ± 3.19	78.95 ± 2.28	48.03 ± 2.66	4.50 ± 2.26

^aThe experimental conditions are in the following order: voltage, initial distance, flow rate, and nozzle speed.

The morphology and average diameters of electrospun mats and structures are shown in Figure 8c and Figure S17, respectively. The average diameter fluctuated. An increase in voltage was able to increase or decrease the diameter of the fiber.^2,8 The influence of voltage is dependent on the threshold or critical point of voltage varying from condition to condition. As seen in Figure S17a, the average diameter at the lower part of applying 13 kV was a little thinner than at 12 kV; however, the middle and upper parts of 13 kV were increased when the structure was upward, which resulted in a closer deposition area to the nozzle. It causes the overall average diameter of 13 kV to be greater than 12 kV, as seen in Figure 8c. The unstable electrospinning at 12 and 13 kV causes dripping and clogging, resulting in a thicker fiber. When the voltage was increased to reach stable electrospinning, smooth fiber was obtained at 14, 15, and 16 kV. The average diameter of 14 kV has dramatically decreased, becoming the thinnest. Furthermore, only the structure at an applied voltage of 14 kV provided no significant difference in electrospun diameter on the upper, middle, and lower parts. After the voltage was increased to 15 and 16 kV, greater diameters were found. The increased jets share a limited electrical field, resulting in a weakened electrical field for each jet.⁴⁰ An increase in voltage increases the interconnective pore or space between the fibers (Figure 8c). Since the repulsive force of fiber increases with an increase in voltage, at 16 kV, an oily flat was found in the lower parts of structures. It was attributed to increasing jet velocity with an increase in voltage.⁸ When the voltage was decreased to 14 and 15 kV, this characteristic disappeared.

The voltage of 14 kV was chosen to be the optimal condition in the 3D buildup of this PCL solution and used to investigate the nozzle speed further.

3.3.4. Nozzle Speed

The nozzle speed of a nozzle at 1, 2, 3, 4, and 5 mm/s was investigated using the optimal conditions of 12 wt % PCL solution in the 6:1 DCM:DMF solvent and the addition of 10% H₃PO₄ at a flow rate of 4 mL/h, an initial distance of 4 cm, and a voltage of 14 kV, as shown in Figure 9a,b. An increase in the nozzle speed causes the 3D formation to rise faster over time, but it causes lower printing quality, changing from a hollow cylinder to a filled circular. The speed is related to the exact position of the fiber deposition, which results in the final shape. Due to discontinuous deposition at faster speeds, the structure deformed into a filled and slanting structure at speeds of 3, 4, and 5 mm/s. Moreover, it was obvious that the solution dried at the tip at 4 and 5 mm/s, as demonstrated in Figure S18. It was attributed to the fast changes in electrospinning parameters and evaporation. The increased evaporation might be explained by a faster nozzle motion, which allows faster airflow around the solution droplet.

Figure 9.

(a) Top and (b) side views of comparative 3D PCL-electrospun constructs by varying the values of the nozzle speed. The nozzle speed applied to the process at 12 wt % PCL solution concentration in 6:1 DCM:DMF + 10% H₃PO₄ was increased from 1 to 5 mm/s. The 3D PCL structures were formed at a flow rate of 4 mL/h, an initial distance of 4 cm, and a voltage of 14 kV for 30 min. (c) SEM images of the upper part of the electrospun mat and the upper, middle, and lower parts of the electrospun mat and 3D structures. The scale bars indicated in (b) and (c) are 10 mm and 50 μm, respectively.

As shown in Table 6, an increase in the nozzle speed allows for higher structures; however, it also poses a risk of deformation. In the case of slower nozzle speeds, it provides the precise position of deposition; however, the accumulation of incoming fibers at single position increases, which may lead to unstable constructs. The width of deposition increased with an increase in the speed. Nevertheless, the width of deposition decreases when the shape is not controlled, resulting in the fiber accumulating at the center and filling the hollow shape. It corresponds to the C_hh increasing with an increase in speed, and the wall thickness increases until no wall thickness occurs. The increase in nozzle speed results in decreased printing quality for hollow cylinders. Applying the speed of 1–2 mm/s, the average fiber diameters remained within a similar range. Larger fiber diameters were found at the top part of the 2 mm/s sample, as seen in Figure S19, whereas the lower and middle parts’ fiber diameters remained comparable to the 1 mm/s sample’s fiber diameter. It was attributed to the higher structures, which allowed the top to be closer to the nozzle. The nozzle speed in these ranges did not have a direct effect on the elongation and drying of the jet. However, when the speed exceeds 2 mm/s, the average diameter of the fiber increases according to the speed. This could be attributed to the drying of the solution at the nozzle that occurred during the experiment. It might be the result of solution drying when the speed increased.

Table 6. Summary of the Overall Average Diameter of Fiber, Height, C_hh, Width of Deposition, D_out, and Wall Thickness of 3D Structures Obtained from Various Moving Speeds of the Nozzle, Including 1, 2, 3, 4, and 5 mm/s^a.

experimental conditions (kV-cm-mL/h-mm/s)	fiber diameter (mm)	height (mm)	Chh (mm)	width of deposition (mm)	Dout (mm)	wall thickness (mm)
14–4–4–1	1.45 ± 1.10	16.18 ± 2.58	15.49 ± 4.60	72.93 ± 2.70	43.85 ± 1.58	3.98 ± 1.01
14–4–4–2	1.82 ± 1.24	24.25 ± 2.63	20.92 ± 2.47	78.56 ± 1.88	46.08 ± 0.95	4.30 ± 1.18
14–4–4–3	3.15 ± 2.17	15.75 ± 9.18	20.57 ± 0.81	-	-	10.32 ± 5.08
14–4–4–4	3.04 ± 1.82	2.05 ± 0.77	-	84.83 ± 1.55	-	22.33 ± 3.60
14–4–4–5	4.46 ± 2.51	4.18 ± 1.59	-	76.65 ± 6.01	-	-

^aThe experimental conditions are in the following order: voltage, initial distance, flow rate, and nozzle speed.

3.4. Surface Modification by Using Plasma Treatment

The morphologies of the untreated and plasma-treated electrospun PCL fibers exhibited little difference, as observed in the SEM images (Figure S20). An incremental rise in diameter was shown to coincide with an increase in the power of the plasma treatment. The average diameters of PCL fibers were 0.97 ± 0.39, 1.03 ± 0.57, 1.41 ± 0.82, 1.43 ± 0.74, and 1.28 ± 0.47 μm for untreated fibers and plasma-treated fibers with 10, 30, 50, and 70 W, respectively. In addition, the pore space between the fibers exhibited a greater width when subjected to the power of 30 or 50 W. This is in agreement with the previously reported result.⁴¹ They showed that the use of an O₂ plasma treatment with a power of 30 W for a duration of 10 min resulted in the induced melting of the thinner diameters of PCL nanofibers, leading to an increased pore size of the sample.

The surface modification of the O₂ plasma treatment was confirmed by FTIR spectra (Figure S21), which shows that hydrophilic groups can be introduced on the surface. The main peaks of PCL remain present in the fiber untreated and treated with the plasma treatment. However, only the FTIR spectrum of treated fibers appeared with new peaks at 3000–3400 and 1670 cm^–1 attributed to OH stretching vibration modes, and 1670 cm^–1 may be the bending mode of adsorbed water or the amide I band.^42,43 Moreover, the wettability was investigated to confirm the improvement of hydrophilic properties of PCL-fibrous structures by the customized setup of the contact angle measurement. As shown in Figure S22, it was found that the contact angles of untreated and treated structures with 10 W for 5 min were 99.34 ± 8.63 and 95.71 ± 0.13 degrees, respectively, while the contact angles of plasma-treated structures with the 30, 50, and 70 W for 5 min cannot be measured because of the fast absorption of water droplets into the surface. The time of complete absorption on the surface of PCL-fibrous structures was 21.00 ± 6.08, 8.76 ± 1.15, and 1.63 ± 1.11 s for fibers untreated and treated with the 10 and 30 W conditions, respectively, and less than 1 s for fibers treated with the 50 and 70 W conditions. The hydrophilic property of PCL-electrospun scaffolds was improved with the plasma treatment, which is consistent with previous results reported in the literature.^28,41,44,45

Figure 10 shows the morphology of NIH3T3 cells that were cultivated for 3 days on three different substrates: the glass slide (control) (Figure 10a), untreated electrospun PCL scaffolds (Figure 10b), and plasma-treated electrospun PCL scaffolds (Figure 10c–f), as observed by SEM. Cell adhesion on the surface of plasma-treated fibers, as shown in Figure 10c–f, was more probable compared to the untreated fibers. It was attributed to the improved wettability. However, an increase in plasma treatment power greater than 50W did not enhance NIH3T3 cell adhesion on scaffolds further. The plasma-treated electrospun scaffold subjected to a 30 or 50 W treatment for 5 min provided the optimal cell morphology and adhesion of NIH3T3 cells on the scaffolds (Figure 10d,e). Further investigation is needed to assess the biological difference between the 30 and 50 W conditions. In this work, the condition of a 50 W treatment was selected as the optimal plasma treatment condition for the PCL-electrospun scaffolds. Additionally, the cytotoxicity of PCL scaffolds was tested and proved that the fabricated scaffolds are not toxic on the NIH3T3 cells, as shown in Figure S25.

Figure 10.

SEM images of cell morphology and adhesion on (a) glass slide and 3D PCL-electrospun structures, which were (b) untreated and treated with (c) 10, (d) 30, (e) 50, and (f) 70 W of O₂ plasma treatment for 5 min. The electrospun structures were fabricated by the 3D electrospinning with conditions of 16 kV of voltage, 4 cm of initial distance, and 4 mL/h of solution flow rate. The scale bar is 10 μm.

3.5. Synchrotron Radiation X-ray Tomography Microscopy (SRXTM)

SRXTM is a highly efficient technique for examining the internal structures of a sample. It generates detailed 3D representations of the sample’s volume, enabling us to visualize internal characteristics without causing any damage, and it also has the ability to accommodate large sample sizes. It was used to observe the cell morphology and adhesion within the 3D electrospun scaffolds cultured for 1 and 3 days. It can present a large field of view that allows for clear visualization of cells on the surface of the scaffolds after normalization, reconstruction, and image processing. Since the intensity distribution of the X-ray absorptance of cells and PCL polymer overlaps, a dyeing process is required. OsO₄ was used for dyeing purposes to facilitate the examination with SRXTM and validate the analysis of cell behavior on the electrospun structures.⁴⁶ The distinction between cells and fibers can be determined by analyzing the disparity in the X-ray absorptance intensity. The pristine PCL-electrospun scaffold without undergoing any cell culturing process and the PCL scaffold with the in vitro culture, including the staining process, were examined by using SRXTM. Figure S23 demonstrates that no signals were observed within the intensity range used to identify stained cells, ensuring this procedure for conducting bioassessment within the 3D electrospun scaffolds.

The SEM and SRXTM images of cell morphology and adhesion during 1 and 3 days of culture are shown in Figure 11. Figure 11a shows little cell count on the electrospun surface for 1 day, even though the SRXTM revealed a discernible distribution of cells inside the scaffolds, as observed in Figure 11b,c and Video S2. This result highlights the potential of wide spatial and in-depth observation. The NIH3T3 cell enables growth on the surface of electrospun scaffolds, as shown in Figure 11a,d. In Figure 11b,e, cells attached to the fibers were shown, and the distribution of cells appeared to be random within the scaffolds. After 3 days of cell culture, the SRXTM results significantly ensured the verification of cell penetration, migration, and proliferation within the electrospun structures (Figure 11c,f), corresponding with the observations made by SEM. The SRXTM images were transferred to the quantitative data by measuring the volume of components with a tool of functions in the Drishti software. Since the number of fibers on each scaffold was different, the volume of cells was normalized by comparing with the volume of fibers to assess the cell growth on the scaffolds during 1 and 3 days of the culture. The normalized volumes of cells on the scaffolds after 1 and 3 days of culture were 0.008 and 0.019, respectively. It implied that there was a 2-fold increase in cell growth on the scaffolds according to the increase in culture days. Figure S24 shows the broader view of SEM images of cells after the culture for 3 days, which illustrates this cell behavior almost everywhere and not only at one point. It is in agreement with the SRXTM observation in Figure 11f.

Figure 11.

SEM and SRXTM images of NIH3T3 cells seeded on PCL-fibrous structures for 1 and 3 days of the culture, which are (a) SEM image showing the surface of the cell on the fibrous structures after seeding the cells into the scaffolds for 1 day, (b) SRXTM image of the NIH3T3 cells (red area) seeded into the scaffolds (a gray area) for 1 day, (c) SRXTM image of the NIH3T3 cell distribution after seeding the cell into the scaffold for 1 day, (d) SEM image showing the cell on the fibrous structures after seeding the cells into the scaffolds for 3 days, (e) SRXTM image of the NIH3T3 cell (red area) seeded into the scaffolds (a gray area) for 3 days, and (f) SRXTM image of the NIH3T3 cell distribution after seeding the cell into the scaffold for 3 days.

4. Conclusion

3D electrospinning, a combination of electrospinning and 3D printing, has been used to build up macroscopic 3D electrospun structures. It has employed rapid solidification, polarization, and electrostatic induction. The phosphoric acid additive in the PCL solutions is necessary for the 3D buildup. In order to extend the use of 3D electrospinning to various materials, it is necessary to initially evaluate the self-assembly capability of the fibers. This evaluation should be conducted by subjecting the solution to the highest flow rate possible over a certain distance, like 5–10 cm. If the fiber is unable to exhibit self-assembly, it is recommended that the additives. However, the quantity of additives is crucial, as an insufficient amount limits the potential of self-assembled fiber formation, while an excessive amount allows for the presence of beads and cannot control the deposition position. Hence, the need for parameter optimization arises.

The optimal solution and process parameters for the 3D PCL structures were found to be a flow rate of 4 mL/h, an initial distance of 4 cm, a voltage of 14 kV, and a nozzle speed of 1 mm/s. The 3D electrospun macrostructures were built with consistent fiber diameters throughout the upper, middle, and lower parts. The height of the structures rose to 16.18 ± 2.58 mm with a C_hh of 15.49 ± 4.60 mm. The deposition width was found to be 72.93 ± 2.70 mm; the D_out was 43.85 ± 1.58 mm, and the wall thickness was 3.98 ± 1.01 mm. The fabricated PCL 3D constructs can retain their final shape for a duration of approximately 4 months. Nevertheless, the composition of the sample has been transformed as a result of the biodegradability of the PCL polymer. The continued existence of nanofiber shapes occurred for a duration of approximately one month. One month later, the fragile structures were observed.

The obtained PCL structures were employed to fabricate scaffolds to conduct a biological assessment of the impact of plasma treatment and see the X-ray tomographic images. We found that the plasma treatment resulted in an increase in the fiber diameter, which was in relation to the power output of the plasma generator. The suitable plasma treatment conditions for treating the PCL scaffolds were found to be 30 and 50 W for 5 min. This treatment led to an enhancement in the wettability of the scaffolds and cell adhesion on the scaffolds. Furthermore, the cell growth and migration of NIH3T3 cells on scaffolds subjected to 50W plasma treatment were verified by SRXTM images. These images provided detailed visualization and the normalized volume of cells within scaffolds, consistent with the SEM images.

5. Challenges

The buildup of 3D electrospun macrostructures by using 3D electrospinning involves many parameters, including solution, electrospinning, speed, height, convergence, wall thickness, and other parameters. Using a positive DC bias on the collector also seems to help with 3D buildup of nanofibers. These parameters vary according to the polymer used; therefore, more studies are required. The incorporation of particles into fibrous structures has not yet been discovered. We had tried to fabricate 3D PCL/hydroxyapatite electrospun structures using 3D electrospinning; however, the collapse of 3D structures after a few minutes, resulting in a 2D mat. It might be due to the additional weight from the hydroxyapatite particles and the insufficient electric force raising itself to achieve self-assembled 3D macrostructures. Therefore, the buildup of composite-based macrostructures via 3D electrospinning is challenging and needs to be investigated further.

Glossary

Abbreviations

2D

two-dimensional

3D

three-dimensional

C_hh

convergence at half height

DCM

dichloromethane

DMEM

Dulbecco’s modified Eagle medium

DMF

dimethylformamide

D_out

outside diameter

FBS

fetal bovine serum

HV

high voltage

PCL

polycaprolactone

PBS

phosphate-buffered saline

SEM

scanning electron microscope

SRXTM

synchrotron radiation X-ray tomographic microscopy

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsbiomaterials.4c00302.

• Additional experiment details about the effect of solution preparation on the 3D buildup, electrospinning parameters on the 3D buildup, plasma treatment on the electrospun fibers, and the confirmation of the staining process for SRXTM (PDF)

• The build-up of the 3D electrospun fiber self-assembly at different time intervals (MP4)

• SRXTM image of the NIH3T3 cell seeded into the scaffolds for 3 days (MP4)

Author Contributions

A.C. and W.N. designed the experiments. A.C. conducted 3D electrospinning. A.C. and O.W. conducted cell culture. A.C., Y.S., and P.P conducted SRXTM. A.C., K.S., and P.J. conducted plasma treatment. A.C. drafted the original manuscript. Y.S., O.W., P.P., S.M., K.S., P.J., N.R., and W.N. reviewed and contributed to the revision of the manuscript drafts. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.