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See a sample of the work done by our amazing collaborators.

High-resolution patterned cellular constructs by droplet-based
3D printing

Bioprinting is an emerging technique for the fabrication of living tissues that allows cells to be arranged in predetermined three-dimensional (3D) architectures. However, to date, there are limited examples of bioprinted constructs containing multiple cell types patterned at high-resolution. Here we present a low-cost process that employs 3D printing of aqueous droplets containing mammalian cells to produce robust, patterned constructs in oil, which were reproducibly transferred to culture medium. Human embryonic kidney (HEK) cells and ovine mesenchymal stem cells (oMSCs) were printed at tissue-relevant densities (107 cells mL−1) and a high droplet resolution of 1 nL. High-resolution 3D geometries were printed with features of ≤200 μm; these included an arborised cell junction, a diagonal-plane junction and an osteochondral interface. The printed cells showed high viability (90% on average) and HEK cells within the printed structures were shown to proliferate under culture conditions. Significantly, a five-week tissue engineering study demonstrated that printed oMSCs could be differentiated down the chondrogenic lineage to generate cartilage-like structures containing type II collagen.

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Engineering a 3D-Bioprinted Model of Human Heart Valve Disease Using Nanoindentation-Based Biomechanics

In calcific aortic valve disease (CAVD), microcalcifications originating from nanoscale calcifying vesicles disrupt the aortic valve (AV) leaflets, which consist of three (biomechanically) distinct layers: the fibrosa, spongiosa, and ventricularis. CAVD has no pharmacotherapy and lacks in vitro models as a result of complex valvular biomechanical features surrounding resident mechanosensitive valvular interstitial cells (VICs). We measured layer-specific mechanical properties of the human AV and engineered a three-dimensional (3D)-bioprinted CAVD model that recapitulates leaflet layer biomechanics for the first time. Human AV leaflet layers were separated by microdissection, and nanoindentation determined layer-specific Young’s moduli. Methacrylated gelatin (GelMA)/methacrylated hyaluronic acid (HAMA) hydrogels were tuned to duplicate layer-specific mechanical characteristics, followed by 3D-printing with encapsulated human VICs. Hydrogels were exposed to osteogenic media (OM) to induce microcalcification, and VIC pathogenesis was assessed by near infrared or immunofluorescence microscopy. Median Young’s moduli of the AV layers were 37.1, 15.4, and 26.9 kPa (fibrosa/spongiosa/ventricularis, respectively). The fibrosa and spongiosa Young’s moduli matched the 3D 5% GelMa/1% HAMA UV-crosslinked hydrogels. OM stimulation of VIC-laden bioprinted hydrogels induced microcalcification without apoptosis. We report the first layer-specific measurements of human AV moduli and a novel 3D-bioprinted CAVD model that potentiates microcalcification by mimicking the native AV mechanical environment. This work sheds light on valvular mechanobiology and could facilitate high-throughput drug-screening in CAVD.

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3D freeform printing of silk fibroin

Freeform fabrication has emerged as a key direction in printing biologically-relevant materials and structures. With this emerging technology, complex structures with microscale resolution can be created in arbitrary geometries and without the limitations found in traditional bottom-up or top-down additive manufacturing methods. Recent advances in freeform printing have used the physical properties of microparticle-based granular gels as a medium for the submerged extrusion of bioinks. However, most of these techniques require post-processing or crosslinking for the removal of the printed structures (Miller et al., 2015; Jin et al., 2016) [1], [2]. In this communication, we introduce a novel method for the one-step gelation of silk fibroin within a suspension of synthetic nanoclay (Laponite) and polyethylene glycol (PEG). Silk fibroin has been used as a biopolymer for bioprinting in several contexts, but chemical or enzymatic additives or bulking agents are needed to stabilize 3D structures. Our method requires no post-processing of printed structures and allows for in situ physical crosslinking of pure aqueous silk fibroin into arbitrary geometries produced through freeform 3D printing.
Statement of Significance

3D bioprinting has emerged as a technology that can produce biologically relevant structures in defined geometries with microscale resolution. Techniques for fabrication of free-standing structures by printing into granular gel media has been demonstrated previously, however, these methods require crosslinking agents and post-processing steps on printed structures. Our method utilizes one-step gelation of silk fibroin within a suspension of synthetic nanoclay (Laponite), with no need for additional crosslinking compounds or post processing of the material. This new method allows for in situ physical crosslinking of pure aqueous silk fibroin into defined geometries produced through freeform 3D printing.

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3D bioprinting for musculoskeletal applications

This review focuses on developments in the field of bioprinting for musculoskeletal tissue engineering, along with discussion on the various approaches for bone, cartilage and connective tissue fabrication. All approaches (cell-laden, cell-free and a combination of both) aim to obtain complex, living tissues able to develop and mature, using the same fundamental technology.

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In Vivo Chondrogenesis in 3D Bioprinted Human Cell-laden Hydrogel Constructs

The three-dimensional (3D) bioprinting technology allows creation of 3D constructs in a layer-by-layer fashion utilizing biologically relevant materials such as biopolymers and cells. The aim of this study is to investigate the use of 3D bioprinting in a clinically relevant setting to evaluate the potential of this technique for in vivo chondrogenesis.

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Parameter optimization for 3D bioprinting of hydrogels

Successful bioprinting of hydrogels relies on geometric accuracy and cell viability, both of which are influenced by a number of variable printing parameters. Despite much research aimed at the resulting quality of bioprinted structures, there is no standard method of comparing bioprint results. In this study, we have developed a simple method of assessing the bioprint results from a range of printing parameters in a standardized manner applicable to extrusion-based bioinks.

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Alginate sulfate-nanocellulose bioinks for cartilage bioprinting
applications

One of the challenges of bioprinting is to identify bioinks which support cell growth, tissue maturation, and ultimately the formation of functional grafts for use in regenerative medicine. The influence of this new biofabrication technology on biology of living cells, however, is still being evaluated. Recently we have identified a mitogenic hydrogel system based on alginate sulfate which potently supports chondrocyte phenotype, but is not printable due to its rheological properties (no yield point). To convert alginate sulfate to a printable bioink, it was combined with nanocellulose, which has been shown to possess very good printability. The alginate sulfate/nanocellulose ink showed good printing properties and the non-printed bioink material promoted cell spreading, proliferation, and collagen II synthesis by the encapsulated cells. When the bioink was printed, the biological performance of the cells was highly dependent on the nozzle geometry. Cell spreading properties were maintained with the lowest extrusion pressure and shear stress. However, extruding the alginate sulfate/nanocellulose bioink and chondrocytes significantly compromised cell proliferation, particularly when using small diameter nozzles and valves.

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Optimization of extrusion based ceramic 3D printing process for complex bony designs

In this study presents materials and design optimization of clinically approved hydroxyapatite (HA) using extrusion based 3D printing process. The effect of various printing parameters including print speed, extrusion pressure, accuracy and infill density to produce defined porous structures is established using various techniques. Particularly Scanning Electron Microscopy, Micro Computed Tomography have been employed to study internal and external accuracy. Mechanical testing was employed to study the effect of porosity on compressive properties of 3D printed structures.

This study shows that, the infill density and shrinkage of 3D printed HA scaffolds post sintering have a linear relationship. Porosity and mechanical strength of 3D printed scaffolds depend on the infill density of the designed CAD file. Tailoring infill density also helps in altering mechanical properties in a predictable manner. Finally, a case study on hydroxyapatite printing of a patient specific bone graft demonstrates the ability of this material and technique to print complex porous structures created on CT-based anatomical bone models and pre-operative 3D planning, providing further promise for custom implant development for complex bony designs.

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Abstract 2072: Combination of CDODA-Me, a glycyrrhetinic acid derivative, and Erlotinib overcomes chemo-resistance in NSCLC PDX spheroids and 3D bio-printed cells

Patient-derived Xenografts (PDXs) are considered as relevant preclinical model for anticancer drug development due to original recapitulation of patient genetic profile, gene expression patterns and tissue histology. In this study, we investigated combination efficacy of CDODA-Me (Methyl 2-cyano-3,11-dioxo-18-olean-1,12-dien-30-oate) and TKI inhibitor Erlotinib (ERL) against Lung NSCLC PDX spheroids and 3D bioprinted PDX cells.

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Combination of CDODA-Me, a glycyrrhetinic acid derivative, and Erlotinib overcomes chemo-resistance in NSCLC PDX spheroids and 3D bio- printed cells

Patient-derived Xenografts (PDXs) are considered as relevant preclinical model for anticancer drug development due to original recapitulation of patient genetic profile, gene expression patterns and tissue histology. In this study, we investigated combination efficacy of CDODA-Me (Methyl 2-cyano-3,11-dioxo-18-olean-1,12-dien-30-oate) and TKI inhibitor Erlotinib (ERL) against Lung NSCLC PDX spheroids and 3D bio-printed PDX cells. NSCLC PDX cells (EGFR T790M mutants) were obtained from Dr. Rishi’s Laboratory. PDX spheroids were grown in DMEM/ F12 media supplemented with L-glutamine, B27 supplement, recombinant human epidermal growth factor (EGF) and recombinant human basic fibroblast growth factor (bFGF). Spheroids were treated with CDODA-Me, ERL alone and in combination. Cell viability was measured by MTT assay. Western blot analysis was used to study the modulation of Bcl-xL, MDR1 and ABCG2 in treated PDX spheroids. For 3D bio-printing of PDX cells, hydrogels were prepared by partial cross-linking of sodium alginate (4.5% w/v) and gelatin (1% w/v) mixture with 40mM CaCl2 solution. PDX Cells were mixed with partially cross-linked hydrogel and printed with Inkredible 3D bio-printer (CELLINK, Sweden). Bio-printed scaffolds were fully cross-linked by 160 mM CaCl2 solution and then incubated overnight with cell culture media. The scaffolds were treated with CDODA-Me and ERL alone and in combination. After 48 h cell viabilities were determined by live/dead assay using fluorescence microscopy.

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