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3D bioengineered skeletal muscle macrotissues are increasingly important for studies of cell biology and development of therapeutics. Tissues derived from immortalized cells obtained from patient samples or from stem cells can be co-cultured with motor-neurons to create models of human neuromuscular junctions in culture. In this study, we present foundational work on 3D cultured muscle ultrastructure, with and without motor neurons, which is enabled by the development of a new co-culture platform. Our results show that tissues from Duchenne muscular dystrophy patients are poorly organized compared to tissues grown from healthy donor and that the presence of motor neurons invariably improves sarcomere organization. Electron micrographs show that in the presence of motor neurons, filament directionality, banding patterns, z-disc continuity and appearance of presumptive SSR and T-tubule profiles all improve in healthy, DMD and iPSC derived muscle tissue. Further work to identify the underlying defects of DMD tissue disorganization and the trophic mechanisms by which motor neurons support muscle are likely to yield potential new therapeutic approaches for treating patients suffering from Duchenne muscular dystrophy.

One significant drawback of existing bioprinted tissues is their lack of shelf-availability caused by complications in both fabrication and storage. Here, we report a cryobioprinting strategy for simultaneously fabricating and storing cell-laden volumetric tissue constructs through seamlessly combining extrusion bioprinting and cryopreservation. The cryobioprinting performance was investigated by designing, fabricating, and storing cell-laden constructs made of our optimized cryoprotective gelatin-based bioinks using a freezing plate with precisely controllable temperature. The in situ freezing process further promoted the printability of cell-laden hydrogel bioinks to achieve freeform structures otherwise inconvenient with direct extrusion bioprinting. The effects of bioink composition on printability and cell viability were evaluated. The functionality of the method was finally investigated using cell differentiation and chick ex ovo assays. The results confirmed the feasibility and efficacy of cryobioprinting as a single-step method for concurrent tissue biofabrication and storage.

Multi-material and multilayered micro- and nanostructures are prominently featured in nature and engineering and are recognized by their remarkable properties. Unfortunately, the fabrication of micro- and nanostructured materials through conventional processes is challenging and costly. Herein, we introduce a high-throughput, continuous, and versatile strategy for the fabrication of polymer fibers with complex multilayered nanostructures. Chaotic electrospinning (ChE) is based on the coupling of continuous chaotic printing (CCP) and electrospinning, which produces fibers with an internal multi-material microstructure. When a CCP printhead is used as an electrospinning nozzle, the diameter of the fibers is further scaled down by 3 orders of magnitude while preserving their internal structure. ChE enables the use of various polymer inks for the creation of nanofibers with a customizable number of internal nanolayers. Our results showcase the versatility and tunability of ChE to fabricate multilayered structures at the nanoscale at high throughput. We apply ChE to the synthesis of unique carbon textile electrodes composed of nanofibers with striations carved into their surface at regular intervals. These striated carbon electrodes with high surface areas exhibit 3- to 4-fold increases in specific capacitance compared to regular carbon nanofibers; ChE holds great promise for the cost-effective fabrication of electrodes for supercapacitors and other applications.

Paclitaxel is a commonly used drug in the medical field because of its strong anticancer effect. However, it may produce relatively severe side effects (i.e., allergic reactions). A major characteristic of paclitaxel is low solubility in water. Special solvents are used for dissolving paclitaxel and preparing the paclitaxel drugs, while the solvents themselves will cause certain effects. Polyoxyethylene castor oil, for example, can cause severe allergic reactions in some people, and the clinical use is limited. In this study, we developed a new Paclitaxel/Poly-L-Lactic Acid (PLLA) nanoparticle drug, which is greatly soluble in water, and carried out in vitro drug sustained release research on it and the original paclitaxel drug. However, because the traditional polymer drug carrier usually uses dialysis bag and thermostatic oscillation system to measure the drug release degree in vitro, the results obtained are greatly different from the actual drug release results in human body. Therefore, this paper adopts the microfluidic chip we previously developed to mimic the human blood vessels microenvironment to study the sustained-release of Paclitaxel/PLLA nanoparticles to make the results closer to the release value in human body. The experimental results showed that compared with the original paclitaxel drug, Paclitaxel/PLLA nanoparticles have a long-sustained release time and a slow drug release, realizing the sustained low-dose release of paclitaxel, a cell cycle-specific anticancer drug, and provided certain reference significance and theoretical basis for the research and development of anticancer drugs.

Suture is an important part of surgical operation, and closure of the wound associated with this procedure continuous to be a challenge in postoperative care. Currently, oxidized regenerated cellulose (ORC) is widely used in the absorption of hemostatic materials. However, there is no ORC medical suture product in the market. The objective of this article was to prepare novel braided sutures by TEMPO-mediated oxidation regenerated cellulose (TORC) to achieve a suturable material with biodegradability and ideal mechanical properties. Regenerated cellulose (RC) strands were made into sutures on a circular braiding machine, and TEMPO-mediated oxidation treatment was introduced alternatively after braiding. The RC sutures under different oxidation time were characterized by ATR-FTIR, electrical conductivity, XRD analysis, physical properties and in vitro degradation property. We further demonstrate that the RC sutures were oxidized and formed the carboxylic (-COOH) functional group. With the extension of oxidation duration, the carboxyl content in TORC sutures increased gradually from 5.1 to 10.4% and the strength, weight, and diameter of TORC sutures decreased gradually. Moreover, we proved that the knot-pull strength of TORC-45 declined by 77.8% after 28 days, thus this sutures fulfilled U.S. Pharmacopeia requirement of knot-pull strength. We have shown that TEMPO oxidation reaction significantly promoted the degradation of TORC sutures. Overall, TORC sutures were successfully produced with favorable biodegradability, revealing potential prospects of clinical applications.

The biofabrication of living constructs containing hollow channels is critical for manufacturing thick tissues. However, current technologies are limited in their effectiveness in the fabrication of channels with diameters smaller than hundreds of micrometers. It is demonstrated that the co-extrusion of cell-laden hydrogels and sacrificial materials through printheads containing Kenics static mixing elements enables the continuous and one-step fabrication of thin hydrogel filaments (1 mm in diameter) containing dozens of hollow microchannels with widths as small as a single cell. Pre-vascularized skeletal muscle-like filaments are bioprinted by loading murine myoblasts (C2C12 cells) in gelatin methacryloyl - alginate hydrogels and using hydroxyethyl cellulose as a sacrificial material. Higher viability and metabolic activity are observed in filaments with hollow multi-channels than in solid constructs. The presence of hollow channels promotes the expression of Ki67 (a proliferation biomarker), mitigates the expression of hypoxia-inducible factor 1-alpha , and markedly enhances cell alignment (i.e., 82% of muscle myofibrils aligned (in ±10°) to the main direction of the microchannels after seven days of culture). The emergence of sarcomeric α-actin is verified through immunofluorescence and gene expression. Overall, this work presents an effective and practical tool for the fabrication of pre-vascularized engineered tissues.

ChE is proposed for the creation of multi-material and multilayered nanostructures. This technology allows for the precise, continuous, and deterministic generation of internal nano-striations. The potential of ChE is shown through the fabrication of a nano-striated carbon electrode that shows a significant increase in capacitance. ChE has the potential of creating multifunctional architectures with enhanced properties for numerous applications.

We report a method for expanding microchannel-embedded paper devices using a precisely controlled gas-foaming technique for the generation of volumetric tissue models in vitro. We successfully fabricated hollow, perfusable microchannel patterns contained in a densely entangled network of bacterial cellulose nanofibrils using matrix-assisted sacrificial three-dimensional printing, and demonstrated the maintenance of their structural integrity after gas-foaming-enabled expansion in an aqueous solution of NaBH4. The resulting expanded microchannel-embedded paper devices showed multilayered laminar structures with controllable thicknesses as a function of both NaBH4 concentration and expansion time. With expansion, the thickness and porosity of the bacterial cellulose network were significantly increased. As such, cellular infiltration was promoted comparing to as-prepared, non-expanded devices. This simple technique enables the generation of truly volumetric, cost-effective human-based tissue models, such as vascularized tumor models, for potential applications in preclinical drug screening and personalized therapeutic selection.

This paper introduces the concept of continuous chaotic printing, i.e. the use of chaotic flows for deterministic and continuous extrusion of fibers with internal multilayered micro- or nanostructures. Two free-flowing materials are coextruded through a printhead containing a miniaturized Kenics static mixer (KSM) composed of multiple helicoidal elements. This produces a fiber with a well-defined internal multilayer microarchitecture at high-throughput (>1.0 m min−1). The number of mixing elements and the printhead diameter determine the number and thickness of the internal lamellae, which are generated according to successive bifurcations that yield a vast amount of inter-material surface area (∼102 cm2 cm−3) at high resolution (∼10 µm). This creates structures with extremely high surface area to volume ratio (SAV). Comparison of experimental and computational results demonstrates that continuous chaotic 3D printing is a robust process with predictable output. In an exciting new development, we demonstrate a method for scaling down these microstructures by 3 orders of magnitude, to the nanoscale level (∼150 nm), by feeding the output of a continuous chaotic 3D printhead into an electrospinner. The simplicity and high resolution of continuous chaotic printing strongly supports its potential use in novel applications, including—but not limited to—bioprinting of multi-scale layered biological structures such as bacterial communities, living tissues composed of organized multiple mammalian cell types, and fabrication of smart multi-material and multilayered constructs for biomedical applications.

This paper introduces the concept of continuous chaotic printing, i.e., the use of chaotic flows for deterministic and continuous fabrication of fibers with internal multilayered micro-or nanostructures. Two free-flowing materials are coextruded through a printhead containing a miniaturized Kenics static mixer (KSM) composed of multiple helicoidal elements. This produces a fiber with a well-defined internal multilayer microarchitecture at high speeds (>1.0 m min-1). The number of mixing elements and the printhead diameter determine the number and thickness of the internal lamellae, which are generated according to successive bifurcations that yield a vast amount of inter-material surface area (~102 cm2 cm3) and high resolution features (~10 μm). In an exciting further development, we demonstrate a scale-down of the microstructure by 3 orders of magnitude, to the nanoscale level (~10 nm), by feeding the output of a continuous chaotic 3D printhead into an electrospinner. Comparison of experimental and computational results demonstrates the robust and predictable output and performance of continuous chaotic 3D printing. The simplicity and high resolution of continuous chaotic printing strongly supports its potential use in novel applications, including—but not limited to—bioprinting of multi-scale tissue-like structures, modeling of bacterial communities, and fabrication of smart multi-material and multilayered constructs.