Additive manufacturing (AM) enables the freeform fabrication of complex structures from various build materials. The objective of this study is to develop a novel Laponite nanoclay-enabled "printing-then-solidification" additive manufacturing approach to extrude complex three-dimensional (3D) structures made of various liquid build materials. Laponite, a member of the smectite mineral family, is investigated to serve as a yield-stress support bath material for the extrusion printing of liquid build materials. Using the printing-then-solidification approach, the printed structure remains liquid and retains its shape with the help of the Laponite support bath. Then the completed liquid structures are solidified in situ by applying suitable cross-linking mechanisms. Finally, the solidified structures are harvested from the Laponite nanoclay support bath for any further processing as needed. Due to its chemical and physical stability, liquid build materials with different solidification/curing/gelation mechanisms can be fabricated in the Laponite bath using the printing-then-solidification approach. The feasibility of the proposed Laponite-enabled printing-then-solidification approach is demonstrated by fabricating several complicated structures made of various liquid build materials, including alginate with ionic cross-linking, gelatin with thermal cross-linking, and SU-8 with photo-cross-linking. During gelatin structure printing, living cells are included and the postfabrication cell viability is above 90%.

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Three dimensional (3D) bioprinting technology enables the freeform fabrication of complex constructs from various hydrogels and is receiving increasing attention in tissue engineering. The objective of this study is to develop a novel self-supporting direct hydrogel printing approach to extrude complex 3D hydrogel composite structures in air without the help of a support bath. Laponite, a member of the smectite mineral family, is investigated to serve as an internal scaffold material for the direct printing of hydrogel composite structures in air. In the proposed printing approach, due to its yield-stress property, Laponite nanoclay can be easily extruded through a nozzle as a liquid and self-supported after extrusion as a solid. Its unique crystal structure with positive and negative charges enables it to be mixed with many chemically and physically cross-linked hydrogels, which makes it an ideal internal scaffold material for the fabrication of various hydrogel structures. By mixing Laponite nanoclay with various hydrogel precursors, the hydrogel composites retain their self-supporting capacity and can be printed into 3D structures directly in air and retain their shapes before cross-linking. Then, the whole structures are solidified in situ by applying suitable cross-linking stimuli. The addition of Laponite nanoclay can effectively improve the mechanical and biological properties of hydrogel composites. Specifically, the addition of Laponite nanoclay results in a significant increase in the Young's modulus of each hydrogel-Laponite composite: 1.9-fold increase for the poly(ethylene glycol) diacrylate (PEGDA)-Laponite composite, 7.4-fold increase for the alginate-Laponite composite, and 3.3-fold increase for the gelatin-Laponite composite.

Freeform fabrication of soft structures has been of great interest in recent years. In particular, it is viewed as a critical step toward the grand vision of organ printing--the on-demand design and fabrication of three-dimensional (3D) human organ constructs for implantation and regenerative medicine. The objective of this study is to develop a novel granular gel support material-enabled, two-step gelation-based 'printing-then-gelation' approach to fabricate 3D alginate structures using filament extrusion. Specifically, a granular Carbopol microgel bath holds the ungelled alginate structure being extruded, avoiding the instantaneous gelation of each printed layer as well as resultant surface tension-induced nozzle clogging. Since Carbopol microgels react with multivalent cations, which are needed for alginate crosslinking, gelatin is introduced as a sacrificial material to make an alginate and gelatin bioink for extrusion, which gels thermally (step-one gelation) to initially stabilize the printed structure for removal from Carbopol. Then gelatin is melted and diffused away while alginate is ionically crosslinked in a 37 °C calcium chloride bath (step-two gelation), resulting in an alginate structure. The proposed 'printing-then-gelation' approach works for alginate structure fabrication, and it is also applicable for the printing of cellular constructs and other similar homogeneous soft structures using a two-step or even multi-step approach. The main conclusions are: (1) 0.8% (w/v) Carbopol bath with a neutral pH value may be most suitable for soft structure printing; (2) it is most effective to use a 0.9% (w/v) NaCl solution to facilitate the removal of residual Carbopol; and (3) alginate structures fabricated using the proposed approach demonstrate better mechanical properties than those fabricated using the conventional 'gelation-while-printing' approach.

Three-dimensional (3D) bioprinting, as a freeform biomedical manufacturing approach, has been increasingly adopted for the fabrication of constructs analogous to living tissues. Generally, materials printed during 3D bioprinting are referred as bioinks, which may include living cells, extracellular matrix materials, cell media, and/or other additives. For 3D bioprinting to be an enabling tissue engineering approach, the bioink printability is a critical requirement as tissue constructs must be able to be printed and reproduce the complex micro-architecture of native tissues in vitro in sufficient resolution. The bioink printability is generally characterized in terms of the controllable formation of well-defined droplets/jets/filaments and/or the morphology and shape fidelity of deposited building blocks. This review presents a comprehensive overview of the studies of bioink printability during representative 3D bioprinting processes, including inkjet printing, laser printing, and micro-extrusion, with a focus on the understanding of the underlying physics during the formation of bioink-based features. A detailed discussion is conducted based on the typical time scales and dimensionless quantities for printability evaluation during bioprinting. For inkjet printing, the Z (the inverse of the Ohnesorge number), Weber, and capillary numbers have been employed for the construction of phase diagrams during the printing of Newtonian fluids, while the Weissenberg and Deborah numbers have been utilized during the printing of non-Newtonian bioinks. During laser printing of Newtonian solutions, the jettability can be characterized using the inverse of the Ohnesorge number, while Ohnesorge, elasto-capillary, and Weber numbers have been utilized to construct phase diagrams for typical non-Newtonian bioinks. For micro-extrusion, seven filament types have been identified including three types of well-defined filaments and four types of irregular filaments. During micro-extrusion, the Oldroyd number has been used to characterize the dimensions of the yielded areas of Herschel-Bulkley fluids. Non-ideal jetting behaviors are common during the droplet-based inkjet and laser printing processes due to the local nonuniformity and nonhomogeneity of cell-laden bioinks.

Stimuli-responsive hydrogels and/or composite hydrogels have been of great interest for various printing applications including four-dimensional printing. Although various responsive hydrogels and/or composite hydrogels have been found to respond to given stimuli and change shapes as designed, the fabrication of three-dimensional (3D) structures from such responsive hydrogels is still a challenge due to their poor 3D printability, and most of the responsive material-based patterns are two-dimensional (2D) in nature. In this study, Laponite nanoclay is studied as an effective additive to improve the self-supporting printability of N-isopropylacrylamide (NIPAAm), a thermoresponsive hydrogel precursor while keeping the responsive functionality of NIPAAm. Graphene oxide (GO) is further added as a nanoscale heater, responding to near-infrared radiation. Due to the different shrinking ratios and mechanical properties of the poly( N-isopropylacrylamide) (pNIPAAm)-Laponite and pNIPAAm-Laponite-GO nanocomposite hydrogels, printed 2D patterns deform in a predictable way. In addition, 3D microfluidic valves are directly printed and cured in air, which can effectively control the flow directions in response to different stimuli as validated in a microfluidic system. Because Laponite nanoclay can be mixed with various responsive hydrogel precursors to improve their 3D printability, the proposed Laponite nanoclay-based nanocomposite hydrogels can be further expanded to prepare various 3D printable responsive nanocomposite hydrogels.

Background Cordyceps militaris, an ascomycete caterpillar fungus, has been used as a traditional Chinese medicine for many years owing to its anticancer and immunomodulatory activities. Currently, artificial culturing of this beneficial fungus has been widely used and can meet the market, but systematic molecular studies on the developmental stages of cultured C. militaris at transcriptional and translational levels have not been determined.Methodology/Principal FindingsWe utilized high-throughput Illumina sequencing to obtain the transcriptomes of C. militaris mycelium and fruiting body. All clean reads were mapped to C. militaris genome and most of the reads showed perfect coverage. Alternative splicing and novel transcripts were predicted to enrich the database. Gene expression analysis revealed that 2,113 genes were up-regulated in mycelium and 599 in fruiting body. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to analyze the genes with expression differences. Moreover, the putative cordycepin metabolism difference between different developmental stages was studied. In addition, the proteome data of mycelium and fruiting body were obtained by one-dimensional gel electrophoresis (1-DGE) coupled with nano-electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC-MS/MS). 359 and 214 proteins were detected from mycelium and fruiting body respectively. GO, KEGG and Cluster of Orthologous Groups (COG) analysis were further conducted to better understand their difference. We analyzed the amounts of some noteworthy proteins in these two samples including lectin, superoxide dismutase, glycoside hydrolase and proteins involved in cordycepin metabolism, providing important information for further protein studies.Conclusions/SignificanceThe results reveal the difference in gene expression between the mycelium and fruiting body of artificially cultivated C. militaris by transcriptome and proteome analysis. Our study provides an effective resource for the further developmental and medicinal research of this promising fungus.

Freeform three-dimensional (3D) printing of functional structures from liquid hydrophobic build materials is of great significance and widely used in various fields such as soft robotics and microfluidics. In particular, a yield-stress support bath-enabled 3D-printing methodology has been emerging to fabricate complex 3D structures. Unfortunately, the reported support bath materials are either hydrophobic or not versatile enough for the printing of a wide range of hydrophobic materials. The objective of this study is to propose a fumed silica nanoparticle-based yield-stress suspension as a hydrophobic support bath to enable 3D extrusion printing of various hydrophobic ink materials in a printing-then-solidification fashion. Hydrophobic ink is freeform-deposited in a hydrophobic fumed silica-mineral oil suspension and maintains its shape during printing; it is not cured until the whole structure is complete. Various hydrophobic inks including poly­(dimethylsiloxane) (PDMS), SU-8 resin, and epoxy-based conductive ink are printed into complex 3D structures in the fumed silica-mineral oil bath and then cured using relevant cross-linking mechanisms, even at a temperature as high as 90 °C, to prove the feasibility and versatility of the proposed printing approach. In addition, the deposited feature can easily reach a much better resolution such as 30 μm for PDMS filaments due to the negligible interfacial tension effect.