4.1.1. Alginate

Alginates extracted from seaweeds and algae are composed of β-1,4-linked blocks of β-D-mannuronic acid (M) and its C-5 epimer α-L-guluronic acid [138]. Alginates are widely used polysaccharides for hydrogelation in tissue engineering as they can be gelated easily through the addition of divalent cations [139]. Since alginate could act as a template for binding of manganese ions, the presence of a high concentration of alginate in the electrolytic manganese dioxide altered the morphology from spindle-shaped to cactusshaped [140].

Spatially modulating the mechanical properties in an alginate bioink, 3D printed constructs were postulated to regulate MSCs' fate. Micro-CT-based imaging with 3D bioprinting and bioreactor system were utilized to fabricate 3D human MSCs-laden porous bone-like scaffolds with varying compressive moduli based on two unmodified polymers (alginate and gelatin). Softer scaffolds with low alginate concentration (0.8% alginate, 0.66 ± 0.08 kPa) revealed accelerated and enhanced osteogenic differentiation with upregulated ALP activity than stiffer scaffolds (1.8% alginate, 5.4 ± 1.2 kPa). In the presence of the osteogenic differentiation medium, cells on soft scaffolds exhibited osteoblastic and early osteocyte-related gene expression and showed a 3D cellular network within the mineralized matrix [141].

Increasing alginate molecular weight, as well as increasing the crosslinking ratio, produces a significantly stiffer bioink. Upon bioprinting cylindrical MSCs laden constructs with spatially variable mechanical stiffness from the core to the periphery, more MSCs underwent osteogenic differentiation within the stiffer regions of the printed constructs as evident by increased ALP staining [142]. In contrast to most studies, an investigation demonstrated that, under basal conditions and in the absence of RGD ligands, alginate hydrogel with bimodal molecular weight distribution (50% LMW and 50% HMW) and 1 wt.% polymer concentration of low-stiffness 3D matrices (tan *∂* ≈ 0.4–0.6) provided a permissive environment for human MSCs osteogenic differentiation and expressed high levels of ALP and osteocalcin as compared to the stiffer 2 wt.% alginate hydrogel with the presence of RGD ligands [143].
