3D Near-Field Electrospinning of Biomaterial Microfibers with Potential for Blended Microfiber-Cell Loaded Gel Composite Structures

This paper describes the development of a novel low-cost and efficient method, 3D near-field electrospinning, to fabricate high-resolution, and repeatable three-dimensional polymeric fiber patterns on nonconductive materials with potential use in tissue engineering. This technology is based on readily available hobbyist grade 3D printers. The result is exquisite control of the deposition of single fibers in an automated manner. Additionally, we demonstrate the fabrication of various fiber patterns, which are subsequently translated to unique cellular patterns. Finally, we printed poly(methyl methacrylate) fibers within 3D collagen gels loaded with cells to introduce anisotropic properties of polymeric fibers within the cell-loaded gels.

Tissue engineering and regenerative medicine are emerging fields with potential to revolutionize modern medicine. Together, the fields of tissue engineering and regenerative medicine, seek to apply stem cells, biomaterials and growth factors either alone or in combination to generate or regenerate damaged and diseased tissues and organs.^[1–3] One of the key elements in the fields of tissue engineering and regenerative medicine is assembling a well-designed scaffold.^[4] However, a scaffold providing an environment that fully resembles the natural extracellular matrix (ECM) remains as a challenge.^[5] Researchers have proposed several different biomaterial strategies, such as, cell-laden hydrogels,^[6] bio-printed biodegradable polymers,^[7] and polymeric nano- and microfibers^{[8, 9]} to create three-dimensional scaffolds with potential for mimicking human tissues or organs. Among these methods, polymeric nano- and microfibers have shown immense potential due to their distinctive properties.^[10] Specifically, polymeric fibers have the ability to create porous three-dimensional platforms resembling the fibrous structure of natural extracellular matrix (ECM). These porous fiber structures have demonstrated an ability to directly regulate many cellular processes, such as, proliferation and differentiation.^[11–13] There are several methods of making polymeric fibers such as melt electrospinning,^{[14, 15]} phase separation,^[16] self-assembly,^[17] and conventional electrospinning.^{[18, 19]} Among all these methods, conventional electrospinning provides a versatile, inexpensive and straightforward electrohydrodynamic process to deposit organic and inorganic nano and micron scale fiber scaffolds with great throughput.^[20–22] To date electrospinning has experienced at least three major stages of development to overcome the disadvantages of earlier stages. These three stages are: conventional electrospinning, field induced electrospinning, and near-field electrospinning. Conventional electrospinning has been widely employed to deposit random fibers as a 3D network with an extensive use in tissue engineering.^[20] However, conventional electrospinning techniques suffer from an unorganized network formation of nanofibers generated by the chaotic whipping of liquid jets,^[23] which leads to difficulties in precisely regulating the orientation of deposited fibers.^[24] Field induced electrospinning has been developed to control deposited fiber alignments through modifying magnetic and electrical fields.^[25] However, this method is not capable of creating precise and organized patterns of fibers. There have been several other attempts focused on applying physical forces to improve the fiber formation and alignments, such as rotational mechanical mandrels.^[26] However, these methods are still not capable of controlling the precise direction of each single fiber. More recently, near-field electrospinning has been introduced as a powerful method to form highly controllable patterns of electrospun fibers on flat surfaces.^[27–29] In near-field electrospinning the distance between needle and collector is very short (500 µm – 3 mm). This relative short distance prevents the bending instability and splitting in electrospun fibers.^[30] Therefore, near-field electrospinning provides the ability to write fibers directly in a highly precise manner. For example, Chang et al. have deposited various complex nanofiber patterns on large and flat areas such as grids or circular arrays via near-field electrospinning.^[31] One of the problems associated with near-field electrospinning systems is that the majority of these systems use complex and expensive multi-axis microscope stages to control the alignment of the fibers in mainly X-Y directions.^[32] Additionally, although near-field electrospinning shows several advantages over conventional and field induced electrospinning, there are still other limitations such as low assembly efficiency, sophisticated set up, limited to forming 2D planar fiber structures and limited patterns. These challenges motivated us to develop an inexpensive platform capable of forming three dimensional, highly precise and reproducible fiber structures through uniting 3D printing technology and near-field electrospinning. 3D printing or additive manufacturing technologies have been subjects of intense study in the field of biomedical engineering to assemble artificial organ structures out of living material, typically referred to as bioprinting.^[7] Bioprinting has the potential to revolutionize the power of medical interventions.^[33] Current technology can create products despite complexity in shape with a resolution of close to 100 µm.^[34] 3D printing also offers several other advantages such as fast processing, minimal loss of raw material as waste, and comparably cost-efficient equipment.^[35] Using these concepts in nano- and microscale fabrication provides the potential to combine near-field electrospinning’s benefits with 3D printing.

In the present work, we report an original automated 3D near-field electrospinning (3DNFES) printer for fabrication of controlled polymer fibers (Figure 1a). Multilayers of highly aligned and patterned polymeric fibers poly(methyl methacrylate) (PMMA) were spun on non-conductive surfaces using 3DNFES. We further demonstrated that we could grow cells on printed fiber patterns and as a proof of concept; we highlight the feasibility of deposition of patterned PMMA fibers within cell-loaded collagen gels. In the following section, we explain the processing details of 3DNFES and report our observations of the resulting patterned fibers with or without the presence of the cells.

The schematic of the custom electrospinning system is illustrated in Figure 1a with the detailed printing process provided in the supplementary information. The 3DNFES system was built through modifying a commercial 3D printer (Markerfarm, Utah, USA), specifically, the printer was modified by replacing the extruder nozzle and heated bed with a custom syringe holder and metal plate. In order to produce the polymer fibers using 3DNFES, 20%(wt/vol) PMMA in Nitromethane was electrospun on non-conductive glass surfaces with the following parameters: flow rate 50 µm h⁻¹, voltage 1.6 kV, spinneret-to-collector 2 mm, and printer speed of 60 mm s⁻¹ (data for 20%(wt/vol) poly(ε-caprolactone) (PCL) in acetone and acetic acid (vol₁/vol₂, 80/20) presented in supplementary data, Figure S4 – S6. In 3DNFES the spinneret-to-collector distance is relatively short, which provides the ability to print precise and reproducible polymer fibers on non-conductive materials such as glass slides, polymer coated glass slides and hydrogel blocks. This capability provides immense potential through eliminating the need of conductive substrate, which is a major drawback for the use of conventional electrospinning and relevant techniques in tissue engineering applications.

To evaluate the 3DNFES process we began by printing some test patterns. Figure 1f displays a crisscrossed pattern of PMMA fibers (1cm × 1cm) on the surface of a glass slide and demonstrates the controllability and continuity of fibers spun by 3DNFES. It is worth mentioning that treatment of glass surfaces with air-plasma noticeably helps the fiber printing uniformity. Fibers made by 3DNFES are relatively uniform in size with a distribution of 1.86 ± 0.41 µm (Figure 1g). Additionally, the 3D printer platform enables the ability to move in X, Y and Z directions in a layer-by-layer style. In the 3DNFES system, the syringe moves in the Y – Z directions, and the collector moves in the X direction. To evaluate the ability of the 3DNFES system to generate patterns, we began with a simple grid, Figure 1h. Next, we investigated the reproducibility in deposition of these simple grid patterns by expanding to multiple fiber layers (Figure 1i – 1k). Scanning electron microscope (SEM) images on Figure 1i – 1k display 2, 3 and 4 layers of PMMA fibers, respectively, constructed on top of each other on a non-conductive glass slide; pseudo color-coded was added for better illustration. Depositing fibers as multilayered patterns is particularly attractive in areas such as, microelectromechanical systems (MEMS), biosensors and flexible electronics.^[36]

Another advantage of 3DNFES is that the diameter of fibers can be adjusted by adjusting operating parameters involved, which is similar to conventional electrospinning.^[37] However, in 3DNFES, in addition to electrospinning parameters, 3D printing parameters such as stage displacement speed and filling capacity of the patterns can play a role in changing fiber shape and fiber density, respectively. We systematically investigated the effects of NFES parameters (polymer concentrations, flow rate, voltage, spinneret-to-collector distance and 3D printing stage displacement speed) on the size and shape of PMMA microfibers. First, we varied the concentration of PMMA in nitromethane from 16 to 24 % (W/V) and observed as the PMMA concentration increased the polymer fiber diameter is increased as well from 1.86 ± 0.41 to 4.73 ± 1.40 µm corresponding to concentrations from 20 to 26 % (W/V, PMMA/nitromethane) while other parameters were kept fix at spinneret-to-collector distance of 2 mm, voltage of 1.6 kV, flowrate of 50 µL h⁻¹ and stage displacement speed of 60 mm s⁻¹, Figure 2a. 20 % PMMA (W/V PMMA/nitromethane) was selected as an optimum concentration throughout the study, since lower concentrations reduced the ability to form continuous fibers due to the low viscosity of the polymer solution, whereas, higher concentrations resulted in significant increases in fiber diameter. Additionally, the 20% PMMA (W/V PMMA/nitromethane) was capable of deposition of fibers on non-conductive gels and glass slide substrates. Next, we studied the influence of flowrate on fiber diameter. Figure 2b illustrates the variation in size of microfiber diameter for the three different flow rates of 50, 100 and 150 µL h⁻¹ at a concentration of 20% PMMA (W/V PMMA/nitromethane). As the flow rate increased from 50 to 150 µL h⁻¹, microfibers grew in diameter from 1.86 ± 0.41 to 3.96 ± 1.31 µm. This increase in fiber diameter is significant even for slight changes of flowrate (50 to 100 µL h⁻¹). For flowrates less than 50 µL h⁻¹ the fibers transition to a series of beads. After investigating the effect of flowrate on fiber size, we evaluated the spinneret-to-collector distance as another key element evolved on changes in fiber diameter. The commercial software, Repetier, was used to control spinneret-to-collector distance. As shown in Figure 2c when the spinneret-to-collector distance is increased, fiber diameters are decreased. Fiber diameters significantly decreased from 3.81 ± 0.89 to 1.54 ± 0.42 µm when spinneret-to-collector distance increased from 1.6 to 2.4 mm. This decrease in fiber diameter is due to increased stretching of the fibers while they travel from spinneret to the collector. The thicker fibers found in the lower spinneret-to-collector distance were non-continuous as a result of identical volumes of the polymer solution. 2 mm was selected as our optimum parameter moving forward based on the production of continuous fibers on our non-conductive substrates. As for the effect of voltage involvement on fiber diameter, we observed by increasing the voltage, fiber diameter also increased (Figure 2d). This observation is contrary with the conventional electrospinning (far-distance electrospinning).^[37] In conventional electrospinning, increasing the voltage results in growth on bending instability and further stretches the fibers, which eventually leads to the formation of thinner fibers. However, in 3DNFES we do not experience the bending instability as a result of short spinneret-to-collector distance, which increasing voltage results in formation of thicker fibers potentially through increasing the rate of material being extruded. This increase in fiber diameter caused the formation of non-continuous and non-uniform fiber patterns. Finally, we investigated the role of stage displacement speed on fiber morphology. In 3DNFES, it is fairly easily to control the stage displacement speed of the 3D printing using the Repetier software. Figure 2e displays the effect of changes on the stage displacement speed (from 10 to 60 mm s⁻¹) on fiber morphologies. At lower speeds, fiber jets are affected by the repulsive force of the charges on the spinneret. These repulsive forces cause oscillatory motions, which lead to wavy and even woven patterned fibers.^[38] The ability to write wavy fibers has potential in several micro devices such as stretchable energy harvesters,^{[39, 40]} stretchable strain sensors,^[41] photonics, electronics, and micromechanics.^[42] Further increasing the stage displacement speed allows the drag force to straighten the fiber and once the stage displacement speed reaches 60 mm s⁻¹ (and higher) fibers were observed to be straight. After evaluating all the parameters, we selected the following optimal parameters: concentration of 20% (W/V PMMA/Nitromethane), flow rate of 50 µL h⁻¹, voltage of 1.6 kV, spinneret-to-collector distance 2 mm, and stage displacement speed of 60 mm s⁻¹.

Another unique advantage of 3DNFES is the ability to form curved fibers when the spinner changes direction. In conventional near-field electrospinning, when the collector stops at the corner especially once the direction of the pattern changes, it results in wavy and woven fibers at the corners.^[43] This happens due to the nature of the motorized stage in which controlling the speed in X and Y directions especially at the edges of patterns are quite difficult. Previously, it was shown that when the collector stopped at the corner (0.2 s), extensive amounts of fibers were created at the corners.^[43] Since 3DNFES utilizes a 3D printer platform we can easily control the stage displacement speed (including speed around the corner and edges) and overcome the pitfalls with near-field electrospinning to achieve more precise, controlled and smoother edge as it is shown in Figure 2f. Finally, we also showed that by adjusting printer filling capacity in the software (from 1% to 9%) we can create sparse and dense fibers in a single run (Figure S1c).