# 3D Printing Applied to Tissue Engineered Vascular grafts
## 3 Three-Dimensional Printing Strategies for TEVG
### Bioprinting
The CaCl\({}_{2}\) solution in the bath had two functions: A cross-linking agent for the alginate, and a support material for printing of the overhanging region, as its density is very close to that of cross-linked alginate. In this study, particular attention was given to the shape-fidelity of the engineered model, and a heuristic approach was implemented to improve it. The modification of the printing trajectories along the  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Manufacturing** & **Accellular** & **Bioink** & **Bioprinted Cells** & **Dimensions** & **Tubes** & **Reference** \\
**Process** & **Materials** & & & & & \\ \hline \multirow{4}{*}{**Extrusion**} & \multirow{2}{*}{Agarose (mold)} & \multirow{2}{*}{-} & HUVSMC, HSF & D: 0.9–2.5 mm & \multirow{2}{*}{B} & \multirow{2}{*}{[72]} \\  & & & SMC (A) & L: 7–10 cm & & \\ \cline{2-6}  & \multirow{2}{*}{
\begin{tabular}{c} NovoGel\({}^{\text{\text{\text{\text{\text{\text{\text{\text{\text{\text{\text{\text{\text{\text{\text{\text{\text{\text{\texttexttext{{        }}}}}}}  
circumferential and axial directions improved the shape-fidelity of tubular structures with bifurcations. The cell viability of the construct was 92.4% immediately after printing and 90.8% after 24 h.  
Furthermore, a photosensitive bioink, composed of sodium alginate blended with gelatin methacryloyl (GelMA) and 4-arm poly (-ethylene glycol)-tetra-acrylate (PESTA), was printed by coaxial extrusion to form perfusable vascular constructs [26]. Upon coaxial extrusion, the hydrogel was first ionically cross-linked by CaCl\({}_{2}\), followed by UV cross-linking and dissolution of the alginate to add porosity to the hollow tube wall. Four-armed PEGTA, as compared to the more common 2-armed PEG, allowed a higher cross-linking density, leading to increased mechanical strength while retaining porosity. Multiple multilayered coaxial nozzles were designed for continuous generation of perfusable constructs with hollow interiors and diameters up to 1.6 mm in a single step process. The direct deposition of the blended photosensitive bioink supported the proliferation and early maturation of vascular cells while remaining printable in a complex 3D vasculature. MSCs and HUVECs were encapsulated together in the bioink, and cultured for up to 21 days using a perfusion bioreactor. Over time, cell migration, spreading, proliferation, as well as MSCs differentiation in SMCs was observed. However, in the meantime, the mechanical properties of the construct deteriorated, due to the degradation of GelMA by the cells. _Fibrin, collagen and gelatin_  
Another interesting hydrogel, fibrin, is derived from the reaction of the enzyme thrombin on the glycoprotein fibrinogen, both extracted from mammalian plasma. _In vivo,_ it combines with platelets to clot wounds in blood vessels. The combination of the two components required for its polymerization makes it well suited for inkjet bioprinting. The main drawbacks of fibrin are its degradation time, which is quick and thus not ideal for the long-term culture of cells, and the difficulty controlling its enzymatic polymerization kinetics. [82; 83]. Human aortic smooth muscle cells (HASMCs) were seeded on fibrin hydrogel to self-organize into a tubular form resembling a natural artery ring [48]. The gel, aided by the innate contractile properties of the SMCs, migrated towards the center post insert, creating a tissue ring of SMCs. These rings were then stacked into the final tubular construct with an inner diameter of 5 mm. Contractility of the construct was observed after 4 days, and mechanical properties were measured after 3 days. Its ultimate circumferential tensile strength was measured to be 191 kPa, which is 3 orders of magnitude lower than that of a natural tunica media layer of a common iliac artery (188 MPa), but was still high enough to allow for an easy manipulation of the construct. Collagen, a well-known protein, is the main component of natural ECM in mammals.