# 3D Printing Applied to Tissue Engineered Vascular grafts
## 3 Three-Dimensional Printing Strategies for TEVG
### Bioprinting
Most hydrogels use type I collagen extracted from rat-tail tendon, porcine skin, and bovine skin. Collagen fibrils self-assemble into bundled fibers at neutral pH. In the presence of water-based solvents, these fibers interact and form a hydrogel. The final properties of the hydrogel depend on the collagen source and polymerization kinetics [84]. Furthermore, the mechanical properties of the collagen fibers can be improved by introducing chemical cross-linkers. The enzymatic degradation of collagen is mediated by the metalloproteinase, secreted by the cells. Gelatin is a biodegradable collagen-based hydrogel that undergoes thermoreversible gelation when its temperature drops below its gel point. The addition of methacrylate groups and photoinitiators makes gelatin photopolymerizable. It then cross-links when exposed to light irradiation to form a hydrogel, with mechanical properties tunable through the ratio of the photo-cross-linker present in the pre-polymer solution [85]. Recently, [PERSON] et al. used mechanical microvalve inkjet to generate in vitro blood vessel models in a bioreactor directly. These TEVGs consisted of a continuous endothelium (HUVEC) imitating the Intima, a human umbilical artery smooth muscle cell (HUASMC) layer mimicking the Media, and a surrounding fibrous and collagenous matrix of normal human dermal fibroblasts (NHDFs) mimicking the Adventitia [38]. HUVECs were suspended in gelatin, HUASMCs in fibrinogen and NHDFs in collagen. The drops were jetted onto the polyether-ether-ketone (PEEK) surface of a chamber that was connected directly to a flow generator. Within the flow bioreactor, the TEVGs were cultivated for up to 3 weeks under physiological conditions. The cell viability was >83% immediately after printing, and the expression of VE-Cadherin, smooth muscle actin, and collagen IV throughout the cultivation period showed that the multilayered wall remained biofunctional. _Poly (ethylene glycol) (PEG)_  
As an alternative to the natural hydrogel, Poly (ethylene glycol) (PEG) is a synthetic hydrogel that is commonly modified with photoinitiators and either diacrylate or methacrylate to become photo cross-linkable [86]. Being synthetic, PEG does not suffer from the batch-to-batch variability and ethical concerns of natural polymers. PEG itself does not allow for cell adhesion and biodegradation, which makes it directly suitable for implantation where the adhesion of tissues must be prevented over long periods [87]. It can be modified with arginine-glycine-aspartic acid (RGD) peptide, found within many adhesion proteins, to allow for cell attachment [88; 89]. Furthermore, by adding degradable cross-linkers and co-polymers during their fabrications, PEG hydrogels can be modified to become bioresorbable [90]. Combining the photo-cross-linking of a self-standing vascular graft with the extrusion of a porous rigid scaffold permitted the creation of a perfusable tissue construct. A biodegradable, photo-cross-linkable poly (ethylene glycol-co-depsipeptide) (PEG-co-PDP) macromere resin was formulated for visible-light SLA fabrication of a cell-laden hydrogel. First, SLA alone was used to manufacture a soft tubular construct out of HUVEC-laden hydrogel resulting in a high shape fidelity [74]. Next, a hybrid bioprinting technique, combining the photo-polymerization of this hydrogel with the extrusion of a solid polymer was developed [75]. A mechanically robust PCL scaffold was extruded simultaneously with the photo-cross-linking of the soft, cell-laden, vascular tubing within the scaffold to form a perfusable, multi-material construct. With this hybrid technique, the mechanical strength of the hydrogel was observed to be as high as that of the surrounding scaffold. 83-95% of cells in the hybrid constructs were alive upon bioprinting and 6 h after. Cell proliferation was observed for up to 10 days in culture. _NovoCGel(r)_  
The inert gel, NovoGel(r) (Organovo), specifically developed for bioprinting, was investigated to create an aorta vascular construct [73]. Based on medical images, [PERSON] et al.