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Various (AB)\(_{n}\) and (ABAC)\(_{n}\) segmented copolymers with hydrophilic and hydrophobic segments are processed via melt electrowriting (MEW). Two different (AB)\(_{n}\) segmented copolymers composed of bisurea segments and hydrophobic poly(dimethyl siloxane) (PDMS) or hydrophilic poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide) (PPO-PEG-PPO) segments, while the amphiphilic (ABAC)\(_{n}\) segmented copolymers consist of bisurea segments in the combination of hydrophobic PDMS segments and hydrophilic PPO-PEG-PPO segments with different ratios, are explored. All copolymer compositions are processed using the same conditions, including nozzle temperature, applied voltage, and collector distance, while changes in applied pressure and collector speed altered the fiber diameter in the range of 7 and 60 µm. All copolymers showed excellent processability with MEW, well-controlled fiber stacking, and inter-layer bonding. Notably, the surfaces of all four copolymer fibers are very smooth when visualized using scanning electron microscopy. However, the fibers show different roughness demonstrated with atomic force microscopy. The non-cytotoxic copolymers increased L929 fibroblast attachment with increasing PDMS content while the different copolymer compositions result in a spectrum of physical properties.
Additive manufacturing of scaffolds with sub-micron filaments via melt electrospinning writing
(2015)
The aim of this study was to explore the lower resolution limits of an electrohydrodynamic process combined with direct writing technology of polymer melts. Termed melt electrospinning writing, filaments are deposited layer-by-layer to produce discrete three-dimensional scaffolds for in vitro research. Through optimization of the parameters (flow rate, spinneret diameter, voltage, collector distance) for poly-ϵ-caprolactone, we could direct-write coherent scaffolds with ultrafine filaments, the smallest being 817 ± 165 nm. These low diameter filaments were deposited to form box-structures with a periodicity of 100.6 ± 5.1 μm and a height of 80 μm (50 stacked filaments; 100 overlap at intersections). We also observed oriented crystalline regions within such ultrafine filaments after annealing at 55 °C. The scaffolds were printed upon NCO-sP(EO-stat-PO)-coated glass slide surfaces and withstood frequent liquid exchanges with negligible scaffold detachment for at least 10 days in vitro.
The skeleton is a preferred homing site for breast cancer metastasis. To date, treatment options for patients with bone metastases are mostly palliative and the disease is still incurable. Indeed, key mechanisms involved in breast cancer osteotropism are still only partially understood due to the lack of suitable animal models to mimic metastasis of human tumor cells to a human bone microenvironment. In the presented study, we investigate the use of a human tissue-engineered bone construct to develop a humanized xenograft model of breast cancer-induced bone metastasis in a murine host. Primary human osteoblastic cell-seeded melt electrospun scaffolds in combination with recombinant human bone morphogenetic protein 7 were implanted subcutaneously in non-obese diabetic/severe combined immunodeficient mice. The tissue-engineered constructs led to the formation of a morphologically intact 'organ' bone incorporating a high amount of mineralized tissue, live osteocytes and bone marrow spaces. The newly formed bone was largely humanized, as indicated by the incorporation of human bone cells and human-derived matrix proteins. After intracardiac injection, the dissemination of luciferase-expressing human breast cancer cell lines to the humanized bone ossicles was detected by bioluminescent imaging. Histological analysis revealed the presence of metastases with clear osteolysis in the newly formed bone. Thus, human tissue-engineered bone constructs can be applied efficiently as a target tissue for human breast cancer cells injected into the blood circulation and replicate the osteolytic phenotype associated with breast cancer-induced bone lesions. In conclusion, we have developed an appropriate model for investigation of species-specific mechanisms of human breast cancer-related bone metastasis in vivo.
3D cell cultures allow a better mimicry of the biological and mechanical environment of cells in vivo compared to 2D cultures. However, 3D cell cultures have been challenging for ultrasoft tissues such as the brain. The present study uses a microfiber reinforcement approach combining mouse primary spinal cord neurons in Matrigel with melt electrowritten (MEW) frames. Within these 3D constructs, neuronal network development is followed for 21 days in vitro. To evaluate neuronal development in 3D constructs, the maturation of inhibitory glycinergic synapses is analyzed using protein expression, the complex mechanical properties by assessing nonlinearity, conditioning, and stress relaxation, and calcium imaging as readouts. Following adaptation to the 3D matrix-frame, mature inhibitory synapse formation is faster than in 2D demonstrated by a steep increase in glycine receptor expression between days 3 and 10. The 3D expression pattern of marker proteins at the inhibitory synapse and the mechanical properties resemble the situation in native spinal cord tissue. Moreover, 3D spinal cord neuronal networks exhibit intensive neuronal activity after 14 days in culture. The spinal cord cell culture model using ultrasoft matrix reinforced by MEW fibers provides a promising tool to study and understand biomechanical mechanisms in health and disease.
Melt electrowriting (MEW) is a high-resolution additive manufacturing technology that balances multiple parametric variables to arrive at a stable fabrication process. The better understanding of this balance is underscored here using high-resolution camera vision of jet stability profiles in different electrical fields. Complementing this visual information are fiber-diameter measurements obtained at precise points, allowing the correlation to electrified jet properties. Two process signatures—the jet angle and for the first time, the Taylor cone area—are monitored and analyzed with a machine vision system, while SEM imaging for diameter measurement correlates real-time information. This information, in turn, allows the detection and correction of fiber pulsing for accurate jet placement on the collector, and the in-process assessment of the fiber diameter. Improved process control is used to successfully fabricate collapsible MEW tubes; structures that require exceptional accuracy and printing stability. Using a precise winding angle of 60° and 300 layers, the resulting 12 mm-thick tubular structures have elastic snap-through instabilities associated with mechanical metamaterials. This study provides a detailed analysis of the fiber pulsing occurrence in MEW and highlights the importance of real-time monitoring of the Taylor cone volume to better understand, control, and predict printing instabilities.
Hydrophilic (AB)\(_{n}\) Segmented Copolymers for Melt Extrusion‐Based Additive Manufacturing
(2021)
Several manufacturing technologies beneficially involve processing from the melt, including extrusion‐based printing, electrospinning, and electrohydrodynamic jetting. In this study, (AB)\(_{n}\) segmented copolymers are tailored for melt‐processing to form physically crosslinked hydrogels after swelling. The copolymers are composed of hydrophilic poly(ethylene glycol)‐based segments and hydrophobic bisurea segments, which form physical crosslinks via hydrogen bonds. The degree of polymerization was adjusted to match the melt viscosity to the different melt‐processing techniques. Using extrusion‐based printing, a width of approximately 260 µm is printed into 3D constructs, with excellent interlayer bonding at fiber junctions, due to hydrogen bonding between the layers. For melt electrospinning, much thinner fibers in the range of about 1–15 µm are obtained and produced in a typical nonwoven morphology. With melt electrowriting, fibers are deposited in a controlled way to well‐defined 3D constructs. In this case, multiple fiber layers fuse together enabling constructs with line width in the range of 70 to 160 µm. If exposed to water the printed constructs swell and form physically crosslinked hydrogels that slowly disintegrate, which is a feature for soluble inks within biofabrication strategies. In this context, cytotoxicity tests confirm the viability of cells and thus demonstrating biocompatibility of this class of copolymers.
There is a specialized niche for the electrohydrodynamic jetting of melts, from biomedical products to filtration and soft matter applications. The next frontier includes optics, microfluidics, flexible electronic devices, and soft network composites in biomaterial science and soft robotics. The recent emphasis on reproducibly direct‐writing continual molten jets has enabled a spectrum of contemporary microscale 3D objects to be fabricated. One strong suit of melt processing is the capacity for the jet to solidify rapidly into a fiber, thus fixing a particular structure into position. The ability to direct‐write complex and multiscaled architectures and structures has greatly contributed to a large number of recent studies, explicitly, toward fiber–hydrogel composites and fugitive inks, and has expanded into several biomedical applications such as cartilage, skin, periosteum, and cardiovascular tissue engineering. Following the footsteps of a publication that summarized melt electrowriting literature up to 2015, the most recent literature from then until now is reviewed to provide a continuous and comprehensive timeline that demonstrates the latest advances as well as new perspectives for this emerging technology.
Macrophages are key players of the innate immune system that can roughly be divided into the pro-inflammatory M1 type and the anti-inflammatory, pro-healing M2 type. While a transient initial pro-inflammatory state is helpful, a prolonged inflammation deteriorates a proper healing and subsequent regeneration. One promising strategy to drive macrophage polarization by biomaterials is precise control over biomaterial geometry. For regenerative approaches, it is of particular interest to identify geometrical parameters that direct human macrophage polarization. For this purpose, we advanced melt electrowriting (MEW) towards the fabrication of fibrous scaffolds with box-shaped pores and precise inter-fiber spacing from 100 μm down to only 40 μm. These scaffolds facilitate primary human macrophage elongation accompanied by differentiation towards the M2 type, which was most pronounced for the smallest pore size of 40 μm. These new findings can be important in helping to design new biomaterials with an enhanced positive impact on tissue regeneration.
Poly(vinylidene fluoride-co-trifluoroethylene) (P(VDF-co-TrFE)) is an electroactive polymer with growing interest for applications in biomedical materials and flexible electronics. In this study, a solvent-free additive manufacturing technique called melt electrowriting (MEW) has been utilized to fabricate well-defined microperiodic structures of the copolymer (P(VDF-co-TrFE)). MEW of the highly viscous polymer melt was initiated using a heated collector at temperatures above 120 °C and required remarkably slow collector speeds below 100 mm min\(^{-1}\). The fiber surface morphology was affected by the collector speed and an increase in β-phase was observed for scaffolds compared to the unprocessed powder. Videography shows vibrations of the P(VDF-co-TrFE) jet previously unseen during MEW, probably due to repeated charge buildup and discharge. Furthermore, piezo-force microscopy measurements demonstrated the electromechanical response of MEW-fabricated fibers. This research therefore achieves the melt electrohydrodynamic processing of fibers with micrometer resolution into defined structures with an important electroactive polymer.
The Multiweek Thermal Stability of Medical-Grade Poly(ε-caprolactone) During Melt Electrowriting
(2022)
Melt electrowriting (MEW) is a high-resolution additive manufacturing technology that places unique constraints on the processing of thermally degradable polymers. With a single nozzle, MEW operates at low throughput and in this study, medical-grade poly(ε-caprolactone) (PCL) is heated for 25 d at three different temperatures (75, 85, and 95 °C), collecting daily samples. There is an initial increase in the fiber diameter and decrease in the jet speed over the first 5 d, then the MEW process remains stable for the 75 and 85 °C groups. When the collector speed is fixed to a value at least 10% above the jet speed, the diameter remains constant for 25 d at 75 °C and only increases with time for 85 and 95 °C. Fiber fusion at increased layer height is observed for 85 and 95 °C, while the surface morphology of single fibers remain similar for all temperatures. The properties of the prints are assessed with no observable changes in the degree of crystallinity or the Young's modulus, while the yield strength decreases in later phases only for 95 °C. After the initial 5-d period, the MEW processing of PCL at 75 °C is extraordinarily stable with overall fiber diameters averaging 13.5 ± 1.0 µm over the entire 25-d period.