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  1. Following the current interest and significant recent advances in three-dimensional printing, the field of tissue engineering is increasingly seeking to adapt this technology for the fabrication of biological tissues, and potentially entire organs, for clinical transplantation. Despite significant demand for vascular grafts for clinical procedures such as coronary bypass surgery, the manufacture of synthetic blood vessels has proved to be problematic. Due to a tendency to cause thrombosis and a lack of growth potential, failure is not uncommon for conduits produced from conventional materials, especially in smaller diameter vessels. As a result, there is great interest in the development of a tissue engineered alternative, and three-dimensional bio-printing may hold the solution. Freeform droplet-based laser bio-printing is an orifice-free printing approach which has been used to generate straight and branched cellular tubes, the fundamental component of engineered blood vessels. As droplet-based laser printing does not require a nozzle, it is particularly well suited to handling the viscous bio-inks often required for tissue engineering purposes without the risk of clogging. By utilising an alginate hydrogel bio-ink capable of carrying a population of living cells, researchers from the University of Florida and Tulane University have printed straight and bifurcated (Y-shaped) tubular structures, demonstrating the promise of 3D-printing technologies for vascular tissue engineering applications. The generation of branched structures is of particular value as these are a fundamental component of native vasculature. Layer-by-layer deposition of droplets of either an 8% alginate solution or a 2% alginate-fibroblast cell suspension was printed following a predesigned pattern. A Z-platform was used to lower each deposited layer, step-wise, into a CaCl2 crosslinking solution to induce gelation of the printed alginate, with each step being the same depth as the height of the previous layer of un-gelled alginate. Initially, acellular straight-line tubes were printed to similar dimensions as human blood vessels, 170 individual layers built over 30 minutes to a height of 5.1mm, to form a tube with an internal diameter of 5mm. Subsequently, 5mm long, 45° Y-shaped tubes were produced, utilising the buoyancy provided by the calcium chloride crosslinking solution to support the formation of overhanging and spanning structures, and taking around two hours to complete. With the printing conditions thus optimised the team then introduced cellular bio-ink into the printing process and reproduced both straight and Y-shaped constructs with an incorporated live cell population. Loading the alginate solutions with cells appeared to disrupt droplet formation to an extent, leading to an increase in the minimum thickness of the vessel walls that could be produced, but the cell viability was considered to be acceptable for a printed bio-ink, and cell numbers were shown to increase over a 24 hour incubation, suggesting that the cells were healthy and proliferative following the bio-printing process. As well as successfully demonstrating the potential of droplet-based laser bio-printing for the tissue engineering of blood vessels and similar tubular structures, this work represents the first example of an overhang being incorporated into a cellular bio-printed construct. Although further development and eventual clinical testing will ultimately be required to determine the suitability of these three dimensional printed blood vessels for therapeutic use, this success brings us a step closer to the use of viable 3D-printed constructs in life-saving vascular graft surgery. Image Credits: http://cellimagelibrary.tumblr.com Biofabrication
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