# 3D Printing Applied to Tissue Engineered Vascular grafts
## 3 Three-Dimensional Printing Strategies for TEVG
### Bioprinting  
Bioprinting enables the fabrication of multi-scale, multi-material, and multicellular tissues with heterogeneous and functional intrinsic structures [70]. With this approach, the engineered tissues present native-like mechanical and structural properties, as well as suitable handling capacities for anastomosis between blood vessels. Bioprinting strategy refers to the incorporation of 3D printed cells in the scaffold. This often implies the functionalization of the scaffold with peptide or protein to achieve tissue maturation. The major remaining challenge is the definition of an optimal bioink and the tailoring of printing techniques allowing high cell viability throughout the manufacturing process. Living cells are printed as a suspension in biomaterials or as aggregates (spheroids or pellets) (Table 2). In the last decade, cell spheroids became popular thanks to novel fabrication and 3D culture techniques. The 3D multicellular arrangement promotes cell-to-cell interaction, leading to an improved tissue-like maturation, as compared to standard 2D cultures. Cell spheroids self-assemble into a fused tissue when placed closely together [71]. Hydrogels are the most popular biomaterials found in bioinks [78; 79]. The water-swollen gels made of 3D hydrophilic polymeric structures are held together by one or multiple forces. These forces can be of different natures, the most common being covalent cross-links, hydrogen bonds, and ionic forces [80]. _Agarose_  
Agarose was one of the first biomaterials investigated for bioprinting. This natural polysaccharide, extracted from reed seaweed, is an inert material with low cell adherence properties. Nevertheless, upon blending with collagen, cell adhesion and viability improved [81]. It is used as a hydrogel in molds for cell aggregation, or as a bioink, with embedded, non-adhering cells [82]. It becomes a gel around 40 \({}^{\circ}\)C, but remains quite viscous even when melted. In 2009, an initial study [72] extruded agarose as a support for different types of cells, including human umbilical vein smooth muscle cells (HUVSMCs) alone, and in combination with a layer of human splenic fibroblasts (HSFs), as well as SMCs alone. The cells were deposited through extrusion, first as a spheroid and later as cell pellets cast in a cylindrical shape. Fusion of the spheroids was observed after 2-4 days, at which point the agarose support was manually removed. Scaffold-free vessels with a 0.9 to 2.5 mm diameter were engineered and maintained for 7 days. _Alginate_  
Alginate is the most popular biocompatible hydrogel used in bioinks. Extruding alginate laden with human glioma U87-MG cells (U87-MGs) into a CaCl2 bath allowed the formation of tubular hydrogel structures [46]. The viability of the cells was 93% immediately after printing and 88% after 11 days of in vitro cell culture. A 2 min post-printing treatment of the model in BaCl\({}_{2}\) solution improved the mechanical stability of the construct, prevented its degradation and increased cell viability. Cell proliferation through the gel and intercellular interaction were observed, implying good porosity of the alginate hydrogel. Based on the same biomaterials, [PERSON] et al. [27] used coaxial extrusion to print alginate laden with HUVECsMCs from the outer nozzle, and cross-linking CaCl\({}_{2}\) solution from the inner nozzle. The cell viability was initially quite low (33%), but increased to 84% after 7 days in culture. Cell activity was confirmed by extra-cellular matrix deposition on both the peripheral and luminal surfaces. Increasing the alginate concentration led to an increased mechanical strength and stability over time, as well as an improved printability. However, the porosity was reduced, leading to a lower cell viability and permeability of the construct. Using a similar approach, vascular-like structures with bifurcations have been successfully printed from jetted alginate, as well as from mouse fibroblast-laden alginate, into a CaCl\({}_{2}\) bath [77].