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International Journal of Bioprinting Internally-crosslinked ADA/Alg/Gel bioinks

Figure 5. Printability characterization of ADA/Alg/Gel hydrogels (A–C) Viscosity curve as a function of the shear rate for ADA/Alg/Gel_50/50/0 (A),
ADA/Alg/Gel_50/40/10 (B), and ADA/Alg/Gel_50/25/25 (C) measured at 2, 10, 20, 30, 45, 60, and 90 min after hydrogel preparation. (D–F) Variations
in fiber diameter over time for ADA/Alg/Gel_50/50/0 (D), ADA/Alg/Gel_50/40/10 (E), and ADA/Alg/Gel_50/25/25 (F) (n = 10). (G) Spreading ratio
over time for ADA/Alg/Gel_50/50/0 (red), ADA/Alg/Gel_50/40/10 (light blue), and ADA/Alg/Gel_50/25/25 (green) (n = 10). *p < 0.05, **p < 0.01, and
****p <0.0001 indicate statistically significant differences with ADA/Alg/Gel_50/50/0. (H) Printability index over time for ADA/Alg/Gel_50/50/0 (red),
ADA/Alg/Gel_50/40/10 (light blue), and ADA/Alg/Gel_50/25/25 (green) (n = 10). (I and J) Optical images of self-standing (I) 3D square samples (10 × 10
2
mm ; five layers) and (J) hollow 3D cylindrical structures (diameter, ∅: 3 mm; 10 layers). Scale bars: 5 mm. Abbreviations: Alg: Alginate; ADA: Alginate
dialdehyde; Gel: Gelatin.

grid structures with an average filament diameter of 465 while 30 min was required for ADA/Alg/Gel_50/25/25
± 80 µm at 10 min and 510.0 ± 85 µm at 20 min. After (Figure 5F). The smallest fiber diameter was achieved
30 min, the average fiber diameter decreased to 330 ± 80 after 45 min for ADA/Alg/Gel_50/40/10 (464 ± 60 µm)
µm, closer to the ideal value of 250 µm (i.e., similar to the and after 60 min for ADA/Alg/Gel_50/25/25 (390 ± 55
nozzle diameter); however, inhomogeneities were observed µm). As for ADA/Alg/Gel_50/50/0 hydrogel, continuous
(Figure 5D). Finally, after 45 min, a continuous grid grid structures could not be obtained even after longer
structure could not be printed due to complete hydrogel durations, i.e., 60 min for ADA/Alg/Gel_50/40/10 hydrogel
crosslinking. The samples containing Gel displayed a and 90 min for ADA/Alg/Gel_50/25/25 hydrogel.
significantly higher fiber size immediately after printing The ADA/Alg/Gel samples demonstrated a similar
due to bioink overflow and delayed crosslinking onset. trend for variations in spreading ratio over time as fiber
Particularly, well-defined grid structures with spaced dimensions (Figure 5G). For ADA/Alg/Gel_50/50/0
individual filaments of ADA/Alg/Gel_50/40/10 could only hydrogel, a spreading ratio between 1.3 and 2.05 was
be printed 20 min after hydrogel preparation (Figure 5E), obtained between 10 and 30 min. The spreading ratio of

Volume 10 Issue 6 (2024) 558 doi: 10.36922/ijb.4014