[n]Cycloparaphenylenes as Compatible Fluorophores for Melt Electrowriting.

Fluorescent probes are an indispensable tool in the realm of bioimaging technologies, providing valuable insights into the assessment of biomaterial integrity and structural properties. However, incorporating fluorophores into scaffolds made from melt electrowriting (MEW) poses a challenge due to the sustained, elevated temperatures that this processing technique requires. In this context, [n]cycloparaphenylenes ([n]CPPs) serve as excellent fluorophores for MEW processing with the additional benefit of customizable emissions profiles with the same excitation wavelength. Three fluorescent blends are used with distinct [n]CPPs with emission wavelengths of either 466, 494, or 533 nm, identifying 0.01 wt% as the preferred concentration. It is discovered that [n]CPPs disperse well within poly(ε-caprolactone) (PCL) and maintain their fluorescence even after a week of continuous heating at 80 °C. The [n]CPP-PCL blends show no cytotoxicity and support counterstaining with commonly used DAPI (Ex/Em: 359 nm/457 nm), rhodamine- (Ex/Em: 542/565 nm), and fluorescein-tagged (Ex/Em: 490/515 nm) phalloidin stains. Using different color [n]CPP-PCL blends, different MEW fibers are sequentially deposited into a semi-woven scaffold and onto a solution electrospun membrane composed of [8]CPP-PCL as a contrasting substrate for the [10]CPP-PCL MEW fibers. In general, [n]CPPs are potent fluorophores for MEW, providing new imaging options for this technology.

Function Follows Form: Oriented Substrate Nanotopography Overrides Neurite-Repulsive Schwann Cell-Astrocyte Barrier Formation in an In Vitro Model of Glial Scarring.

Schwann cell (SC) transplantation represents a promising therapeutic approach for traumatic spinal cord injury but is frustrated by barrier formation, preventing cell migration, and axonal regeneration at the interface between grafted SCs and reactive resident astrocytes (ACs). Although regenerating axons successfully extend into SC grafts, only a few cross the SC-AC interface to re-enter lesioned neuropil. To date, research has focused on identifying and modifying the molecular mechanisms underlying such scarring cell-cell interactions, while the influence of substrate topography remains largely unexplored. Using a recently modified cell confrontation assay to model SC-AC barrier formation in vitro, highly oriented poly(ε-caprolactone) nanofibers were observed to reduce AC reactivity, induce extensive oriented intermingling between SCs and ACs, and ultimately enable substantial neurite outgrowth from the SC compartment into the AC territory. It is anticipated that these findings will have important implications for the future design of biomaterial-based scaffolds for nervous tissue repair.

Electrohydrodynamic 3D Printing of Aqueous Solutions.

Among the various electrohydrodynamic (EHD) processing techniques, electrowriting (EW) produces the most complex 3D structures. Aqueous solution EW similarly retains the potential for additive manufacturing well-resolved 3D structures, while providing new opportunities for processing biologically derived polymers and eschewing organic solvents. However, research on aqueous-based EHD processing is still limited. To summarize the field and advocate for increased use of aqueous bio-based materials, this review summarizes the most significant contributions of aqueous solution processing. Special emphasis has been placed on understanding the effects of different printing parameters, the prospects for 3D processing new materials, and future challenges.

Melt Electrowriting of Nylon-12 Microfibers with an Open-Source 3D Printer.

This study demonstrates how either a heated flat or cylindrical collector enables defect-free melt electrowriting (MEW) of complex geometries from high melting temperature polymers. The open-source "MEWron" printer uses nylon-12 filament and combined with a heated flat or cylindrical collector, produces well-defined fibers with diameters ranging from 33 ± 4 to 95 ± 3 µm. Processing parameters for stable jet formation and minimal defects based on COMSOL thermal modeling for hardware design are optimized. The balance of processing temperature and collector temperature is achieved to achieve auxetic patterns, while showing that annealing nylon-12 tubes significantly alters their mechanical properties. The samples exhibit varied pore sizes and wall thicknesses influenced by jet dynamics and fiber bridging. Tensile testing shows nylon-12 tubes are notably stronger than poly(ε-caprolactone) ones and while annealing has limited impact on tensile strength, yield, and elastic modulus, it dramatically reduces elongation. The equipment described and material used broadens MEW applications for high melting point polymers and highlights the importance of cooling dynamics for reproducible samples.

The Multiweek Thermal Stability of Medical-Grade Poly(ε-caprolactone) During Melt Electrowriting.

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.

An initiator- and catalyst-free hydrogel coating process for 3D printed medical-grade poly(ε-caprolactone).

Additive manufacturing or 3D printing as an umbrella term for various materials processing methods has distinct advantages over many other processing methods, including the ability to generate highly complex shapes and designs. However, the performance of any produced part not only depends on the material used and its shape, but is also critically dependent on its surface properties. Important features, such as wetting or fouling, critically depend mainly on the immediate surface energy. To gain control over the surface chemistry post-processing modifications are generally necessary, since it's not a feature of additive manufacturing. Here, we report on the use of initiator and catalyst-free photografting and photopolymerization for the hydrophilic modification of microfiber scaffolds obtained from hydrophobic medical-grade poly(ε-caprolactone) via melt-electrowriting. Contact angle measurements and Raman spectroscopy confirms the formation of a more hydrophilic coating of poly(2-hydroxyethyl methacrylate). Apart from surface modification, we also observe bulk polymerization, which is expected for this method, and currently limits the controllability of this procedure.

Melt Electrospinning of Nanofibers from Medical-Grade Poly(ε-Caprolactone) with a Modified Nozzle.

Melt electrospun fibers, in general, have larger diameters than normally achieved with solution electrospinning. This study uses a modified nozzle to direct-write melt electrospun medical-grade poly(ε-caprolactone) onto a collector resulting in fibers with the smallest average diameter being 275 ± 86 nm under certain processing conditions. Within a flat-tipped nozzle is a small acupuncture needle positioned so that reduces the flow rate to ≈0.1 µL h-1 and has the sharp tip protruding beyond the nozzle, into the Taylor cone. The investigations indicate that 1-mm needle protrusion coupled with a heating temperature of 120 °C produce the most consistent, small diameter nanofibers. Using different protrusion distances for the acupuncture needle results in an unstable jet that deposited poor quality fibers that, in turn, affects the next adjacent path. The material quality is notably affected by the direct-writing speed, which became unstable above 10 mm min-1 . Coupled with a dual head printer, first melt electrospinning, then melt electrowriting could be performed in a single, automated process for the first time. Overall, the approach used here resulted in some of the smallest melt electrospun fibers reported to date and the smallest diameter fibers from a medical-grade degradable polymer using a melt processing technology.

Hydrogels with Cell Adhesion Peptide-Decorated Channel Walls for Cell Guidance.

A method is reported for making hollow channels within hydrogels decorated with cell-adhesion peptides exclusively at the channel surface. Sacrificial fibers of different diameters are used to introduce channels within poly(ethylene glycol) hydrogels crosslinked with maleimide-thiol chemistry, which are backfilled with a cysteine-containing peptide solution which is conjugated to the lumen with good spatial efficiency. This allows for peptide patterning in only the areas of the hydrogel where they are needed when used as cell-guides, reducing the amount of required peptide 20-fold when compared to bulk functionalization. The power of this approach is highlighted by successfully using these patterned hydrogels without active perfusion to guide fibroblasts and olfactory ensheathing cells-the latter having unique potential in neural repair therapies.

Out-of-Plane 3D-Printed Microfibers Improve the Shear Properties of Hydrogel Composites.

One challenge in biofabrication is to fabricate a matrix that is soft enough to elicit optimal cell behavior while possessing the strength required to withstand the mechanical load that the matrix is subjected to once implanted in the body. Here, melt electrowriting (MEW) is used to direct-write poly(ε-caprolactone) fibers "out-of-plane" by design. These out-of-plane fibers are specifically intended to stabilize an existing structure and subsequently improve the shear modulus of hydrogel-fiber composites. The stabilizing fibers (diameter = 13.3 ± 0.3 µm) are sinusoidally direct-written over an existing MEW wall-like structure (330 µm height). The printed constructs are embedded in different hydrogels (5, 10, and 15 wt% polyacrylamide; 65% poly(2-hydroxyethyl methacrylate) (pHEMA)) and a frequency sweep test (0.05-500 rad s-1 , 0.01% strain, n = 5) is performed to measure the complex shear modulus. For the rheological measurements, stabilizing fibers are deposited with a radial-architecture prior to embedding to correspond to the direction of the stabilizing fibers with the loading of the rheometer. Stabilizing fibers increase the complex shear modulus irrespective of the percentage of gel or crosslinking density. The capacity of MEW to produce well-defined out-of-plane fibers and the ability to increase the shear properties of fiber-reinforced hydrogel composites are highlighted.

Dimension-Based Design of Melt Electrowritten Scaffolds.

The electrohydrodynamic stabilization of direct-written fluid jets is explored to design and manufacture tissue engineering scaffolds based on their desired fiber dimensions. It is demonstrated that melt electrowriting can fabricate a full spectrum of various fibers with discrete diameters (2-50 µm) using a single nozzle. This change in fiber diameter is digitally controlled by combining the mass flow rate to the nozzle with collector speed variations without changing the applied voltage. The greatest spectrum of fiber diameters was achieved by the simultaneous alteration of those parameters during printing. The highest placement accuracy could be achieved when maintaining the collector speed slightly above the critical translation speed. This permits the fabrication of medical-grade poly(ε-caprolactone) into complex multimodal and multiphasic scaffolds, using a single nozzle in a single print. This ability to control fiber diameter during printing opens new design opportunities for accurate scaffold fabrication for biomedical applications.

Spatial Patterning of Hydrogels via 3D Covalent Transfer Stamping from a Fugitive Ink.

In this study, a novel hydrogel patterning approach using 3D covalent transfer stamping from a fugitive ink is introduced. A model transfer molecule (7-diethylamino-3-(4'maleimidylphenyl)-4-methylcoumarin (CPM)) is absorbed into a 3D fugitive ink stamp made from poly(caprolactone), and then encapsulated in a poly(ethylene glycol) hydrogel. As the CPM diffuses from the fugitive ink to the curing hydrogel it is selectively covalently bound at the boundary of the ink and the hydrogel. Removal of the fugitive ink by solvent exchange leads to a negative copy of interconnected channels patterned with the localized transfer of the molecules at the hydrogel interface. The results suggest that small molecules can be patterned in porous hydrogels in a spatially controllable manner and that the transferred amount can be tuned.

Melt Electrowriting of Thermoplastic Elastomers.

Melt electrowriting (MEW), an additive manufacturing process, is established using polycaprolactone as the benchmark material. In this study, a thermoplastic elastomer, namely, poly(urea-siloxane), is synthesized and characterized to identify how different classes of polymers are compatible with MEW. This polyaddition polymer has reversible hydrogen bonding from the melt upon heating/cooling and highly resolved structures are achieved by MEW. The influence of applied voltage, temperature, and feeding pressure on printing outcomes behavior is optimized. Balancing these parameters, highly uniform and smooth-surfaced fibers with diameters ranging from 10 to 20 µm result. The quality of the 3D MEW scaffolds is excellent, with very accurate fiber stacking capacity-up to 50 layers with minimal defects and good fiber fusion between the layers. There is also minimal fiber sagging between the crossover points, which is a characteristic of thicker MEW scaffolds previously reported with other polymers. In summary, poly(urea-siloxane) demonstrates outstanding compatibility with the MEW process and represents a class of polymer-thermoplastic elastomers-that are, until now, untested with this approach.

Selective isolation and characterization of primary cells from normal breast and tumors reveal plasticity of adipose derived stem cells.

BACKGROUND: There is a need to establish more cell lines from breast tumors in contrast to immortalized cell lines from metastatic effusions in order to represent the primary tumor and not principally metastatic biology of breast cancer. This investigation describes the simultaneous isolation, characterization, growth and function of primary mammary epithelial cells (MEC), mesenchymal cells (MES) and adipose derived stem cells (ADSC) from four normal breasts, one inflammatory and one triple-negative ductal breast tumors. METHODS: A total of 17 cell lines were established and gene expression was analyzed for MEC and MES (n = 42) and ADSC (n = 48) and MUC1, pan-KRT, CD90 and GATA-3 by immunofluorescence. DNA fingerprinting to track cell line identity was performed between original primary tissues and isolates. Functional studies included ADSC differentiation, tumor MES and MEC invasion co-cultured with ADSC-conditioned media (CM) and MES adhesion and growth on 3D-printed scaffolds. RESULTS: Comparative analysis showed higher gene expression of EPCAM, CD49f, CDH1 and KRTs for normal MEC lines; MES lines e.g. Vimentin, CD10, ACTA2 and MMP9; and ADSC lines e.g. CD105, CD90, CDH2 and CDH11. Compared to the mean of all four normal breast cell lines, both breast tumor cell lines demonstrated significantly lower ADSC marker gene expression, but higher expression of mesenchymal and invasion gene markers like SNAI1 and MMP2. When compared with four normal ADSC differentiated lineages, both tumor ADSC showed impaired osteogenic and chondrogenic but enhanced adipogenic differentiation and endothelial-like structures, possibly due to high PDGFRB and CD34. Addressing a functional role for overproduction of adipocytes, we initiated 3D-invasion studies including different cell types from the same patient. CM from ADSC differentiating into adipocytes induced tumor MEC 3D-invasion via EMT and amoeboid phenotypes. Normal MES breast cells adhered and proliferated on 3D-printed scaffolds containing 20 fibers, but not on 2.5D-printed scaffolds with single fiber layers, important for tissue engineering. CONCLUSION: Expression analyses confirmed successful simultaneous cell isolations of three different phenotypes from normal and tumor primary breast tissues. Our cell culture studies support that breast-tumor environment differentially regulates tumor ADSC plasticity as well as cell invasion and demonstrates applications for regenerative medicine.

Additive Manufacturing of a Photo-Cross-Linkable Polymer via Direct Melt Electrospinning Writing for Producing High Strength Structures.

Melt electrospinning writing (MEW) is an emerging additive manufacturing technique that enables the design and fabrication of micrometer-thin fibrous scaffolds made of biocompatible and biodegradable polymers. By using a computer-aided deposition process, a unique control over pore size and interconnectivity of the resulting scaffolds is achieved, features highly interesting for tissue engineering applications. However, MEW has been mainly used to process low melting point thermoplastics such as poly(ε-caprolactone). Since this polymer exhibits creep and a reduction in modulus upon hydration, we manufactured scaffolds of poly(L-lactide-co-ε-caprolactone-co-acryloyl carbonate) (poly(LLA-ε-CL-AC)), a photo-cross-linkable and biodegradable polymer, for the first time. We show that the stiffness of the scaffolds increases significantly (up to ∼10-fold) after cross-linking by UV irradiation at room temperature, compared with un-cross-linked microfiber scaffolds. The preservation of stiffness and high average fiber modulus (370 ± 166 MPa) within the cross-linked hydrated scaffolds upon repetitive loading (10% strain at 1 Hz up to 200,000 cycles) suggests that the prepared scaffolds may be of potential interest for soft connective tissue engineering applications. Moreover, the approach can be readily adapted through manipulation of polymer properties and scaffold geometry to prepare structures with mechanical properties suitable for other tissue engineering applications.

Hierarchically Structured Porous Poly(2-oxazoline) Hydrogels.

A new method for fabricating hydrogels with intricate control over hierarchical 3D porosity using microfiber porogens is presented. Melt electrospinning writing of poly(ε-caprolactone) is used to create the sacrificial template leading to hierarchical structuring consisting of pores inside the denser poly(2-oxazoline) hydrogel mesh. This versatile approach provides new opportunities to create well-defined multilevel control over interconnected pores with diameters in the lower micrometer range inside hydrogels with potential applications as cell scaffolds with tunable diffusion and transport of, e.g., nutrients, growth factors or therapeutics.

Why bioprinting in regenerative medicine should adopt a rational technology readiness assessment.

Bioprinting is an annex of additive manufacturing, as defined by the American Society for Testing and Materials (ASTM) and International Organization for Standardization (ISO) standards, characterized by the automated deposition of living cells and biomaterials. The tissue engineering and regenerative medicine (TE&RM) community has eagerly adopted bioprinting, while review articles regularly herald its imminent translation to the clinic as functional tissues and organs. Here we argue that such proclamations are premature and counterproductive; they place emphasis on technological progress while typically ignoring the critical stage-gates that must be passed through to bring a technology to market. We suggest the technology readiness level (TRL) scale as a valuable metric for gauging the relative maturity of a bioprinting technology in relation to how it has passed a series of key milestones. We suggest guidelines for a bioprinting-oriented scale and use this to discuss the state-of-the-art of bioprinting in regenerative medicine (BRM) today. Finally, we make corresponding recommendations for improvements to BRM research that would support its progression to clinical translation.

Materials and Strategies to Enhance Melt Electrowriting Potential.

Melt electrowriting (MEW) is an emerging additive manufacturing (AM) technology that enables the precise deposition of continuous polymeric microfibers, allowing for the creation of high-resolution constructs. In recent years, MEW has undergone a revolution, with the introduction of active properties or additional functionalities through novel polymer processing strategies, the incorporation of functional fillers, postprocessing, or the combination with other techniques. While extensively explored in biomedical applications, MEW's potential in other fields remains untapped. Thus, this review explores MEW's characteristics from a materials science perspective, emphasizing the diverse range of materials and composites processed by this technique and their current and potential applications. Additionally, the prospects offered by postprinting processing techniques are explored, together with the synergy achieved by combining melt electrowriting with other manufacturing methods. By highlighting the untapped potentials of MEW, this review aims to inspire research groups across various fields to leverage this technology for innovative endeavors.

Cryo-Electrohydrodynamic Jetting of Aqueous Silk Fibroin Solutions.

The incorporation of 3D-printing principles with electrohydrodynamic (EHD) jetting provides a harmonious balance between resolution and processing speed, allowing for the creation of high-resolution centimeter-scale constructs. Typically, EHD jetting of polymer melts offers the advantage of rapid solidification, while processing polymer solutions requires solvent evaporation to transition into solid fibers, creating challenges for reliable printing. This study navigates a hybrid approach aimed at minimizing printing instabilities by combining viscous solutions and achieving rapid solidification through freezing. Our method introduces and fully describes a modified open-source 3D printer equipped with a frozen collector that operates at -35 °C. As a proof of concept, highly concentrated silk fibroin aqueous solutions are processed into stable micrometer scale jets, which rapidly solidify upon contact with the frozen collector. This results in the formation of uniform microfibers characterized by an average diameter of 27 ± 5 µm, a textured surface, and porous internal channels. The absence of instabilities and the notably fast direct writing speed of 42 mm·s-1 enable precise, fast, and reliable deposition of these fibers into porous constructs spanning several centimeters. The effectiveness of this approach is demonstrated by the consistent production of biologically relevant scaffolds that can be customized with varying pore sizes and shapes. The achieved degree of control over micrometric jet solidification and deposition dynamics represents a significant advancement in EHD jetting, particularly within the domain of aqueous polymer solutions, offering new opportunities for the development of intricate and functional biological structures.

Electrowriting of SU-8 Microfibers.

As microfiber-based additive manufacturing (AM) technologies, melt electrowriting (MEW) and solution electrowriting (SEW) have demonstrated efficacy with more biomedically relevant materials. By processing SU-8 resin using MEW and SEW techniques, a material with substantially different mechanical, thermal, and optical properties than that typically processed is introduced. SU-8 polymer is temperature sensitive and requires the devising of a specific heating protocol to be properly processed. Smooth-surfaced microfibers resulted from MEW of SU8 for a short period (from 30 to 90 min), which provides the greatest control and, thus, reproducibility of the printed microfibers. This investigation explores various parameters influencing the electrowriting process, printing conditions, and post-processing to optimize the fabrication of intricate 3D structures. This work demonstrates the controlled generation of straight filaments and complex multi-layered architectures, which were characterized by brightfield, darkfield, and scanning electron microscopy (SEM). This research opens new avenues for the design and development of 3D-printed photonic systems by leveraging the properties of SU-8 after both MEW and SEW processing.

Bioassembly of hemoglobin-loaded photopolymerizable spheroids alleviates hypoxia-induced cell death.

The delivery of oxygen within tissue engineered constructs is essential for cell survivability; however, achieving this within larger biofabricated constructs poses a significant challenge. Efforts to overcome this limitation often involve the delivery of synthetic oxygen generating compounds. The application of some of these compounds is problematic for the biofabrication of living tissues due to inherent issues such as cytotoxicity, hyperoxia and limited structural stability due to oxygen inhibition of radical-based crosslinking processes. This study aims to develop an oxygen delivering system relying on natural-derived components which are cytocompatible, allow for photopolymerization and advanced biofabrication processes, and improve cell survivability under hypoxia (1% O2). We explore the binding of human hemoglobin (Hb) as a natural oxygen deposit within photopolymerizable allylated gelatin (GelAGE) hydrogels through the spontaneous complex formation of Hb with negatively charged biomolecules (heparin, hyaluronic acid, and bovine serum albumin). We systematically study the effect of biomolecule inclusion on cytotoxicity, hydrogel network properties, Hb incorporation efficiency, oxygen carrying capacity, cell viability, and compatibility with 3D-bioassembly processes within melt electrowritten (MEW) scaffolds. All biomolecules were successfully incorporated within GelAGE hydrogels, displaying controllable mechanical properties and cytocompatibility. Results demonstrated efficient and tailorable Hb incorporation within GelAGE-Heparin hydrogels. The developed system was compatible with microfluidics and photopolymerization processes, allowing for the production of GelAGE-Heparin-Hb spheres. Hb-loaded spheres were assembled into MEW polycaprolactone scaffolds, significantly increasing the local oxygen levels. Ultimately, cells within Hb-loaded constructs demonstrated good cell survivability under hypoxia. Taken together, we successfully developed a hydrogel system that retains Hb as a natural oxygen deposit post-photopolymerization, protecting Hb from free-radical oxidation while remaining compatible with biofabrication of large constructs. The developed GelAGE-Heparin-Hb system allows for physoxic oxygen delivery and thus possesses a vast potential for use across broad tissue engineering and biofabrication strategies to help eliminate cell death due to hypoxia.