# Use of Anionic Polysaccharides in the Development of 3D Bioprinting Technology
## 3 3D Bioprinting Technology Concept
It is reported that successful fabrication of a cell-laden gel tube is around 400 \(\upmu\)m in diameter (Figure 5a). There is a commercially available bioink named Cellink (Cellink, Gothenburg, Sweden) which is composed of alginate, a cellulose nanofiber-containing polymer with enhanced shear-thinning properties. Due to the shear-thinning property, printed 3D constructs can retain the shape during printing. Post-printing, the constructs are gelated by reacting with an aqueous solution of Ca\({}^{2+}\). A noticeable drawback of sodium alginate for tissue engineering applications is the low cell attachment and characteristic spreading of the resultant hydrogels crosslinked by divalent cations. To address this problem, [PERSON] et al. used an alginate conjugated with cell-adhesive RGD peptide for bioprinting [74]. Another approach in utilizing the hydrogelation feature is a combination with other materials having different properties with alginate such as hydroxyapatite [75] and gelatin [76]. [PERSON] et al. used a bioink containing gelatin, alginate, hydroxyapatite, and cells to print a novel cell-laden composite for bone printing using micro-extrusion bioprinting [76]. In the system, the bioink was first extruded onto a cooling plate for inducing thermal hydrogenation attributed to gelatin. Then, the resultant constructs were immersed in a solution containing Ca\({}^{2+}\) for inducing cross-linking of alginate to improve a long-term structural integrity of the constructs including cells. Alginate has also been used as a component of bioink after being introduced to phenolic hydroxyl moieties that proved to be cross-linkable under mild conditions. In these cases, the biocompatibility and low toxicity would be considered useful. [PERSON] et al. fabricated 3D constructs through micro-extrusion bioprinting using bioink containing photo-cross-linkable and cell-adhesive alginate derivative obtained by introducing both methacrylate and RGD moieties onto the alginate backbone [77]. In the system, hydrogels were obtained through ultraviolet light-irradiation in the presence of a photo initiator. [PERSON] et al. reported stereography bioprinting using alginate derivatives obtained by introducing phenolic hydroxyl moieties through visible light irradiation in the presence of ruthenium II tris(bipyridyl)chloride and sodium persulfate (Figure 5b) [78]. Fibroblast cells enclosed in the hydrogel constructs showed more than 90% viability for seven days [78]. The same alginate derivative possessing phenolic hydroxyl moieties were also used as bioinks of inkjet bioprinting (Figure 5c) [79] and micro-extrusion bioprinting [80]. In these studies, the bioinks were gelated through horseradish peroxidase-catalyzed reaction. The enzyme-mediated hydrogenation was reported as rapid enough to fabricate 3D constructs with the shapes as designed [79; 80]. Hyaluronic acid (HA), one of the most important components of extracellular matrices, is a polysaccharide that has been intensively investigated as a component of bioink due to its excellent biocompatibility and biological properties [81; 82]. Native HA is not gellable under physiological conditions, therefore introduction of cross-linkable moieties is necessary for the use as bioinks to fabricate the 3D constructs containing HA in stable. As the similar manner for alginate described above, HA derivatives possessing introduced methacrylate moieties [83] and phenolic hydroxyl moieties [79; 84] were used in micro-extrusion bioprinting (Figure 5d) [83; 84] and inkjet bioprinting [79]. Considering the various bioactive properties of each polysaccharide, these polysaccharides will be used as components of bioinks through the introduction of crosslink moieties catalyzed by enzymatic means.