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Tan, et al.
modulus reached a plateau. As observed with FG7.5 was simulated with 1 × PBS, which would be an
and FG10, the change in gelatin concentration had important consideration if the samples were used
little effect on the gelation time for FG. However, as cellular scaffolds. We studied the swelling
the shorter gelation time in FG10 (~168 s) than of hydrogels by placing the hydrogel samples
FG7.5 (~174 s) may explain the mild spreading of prepared from FG (FG7.5, FG10, and FG20)
FG7.5 ink at 3 min (Figure 2A), which was not and PG (PG7.5, PG10, and PG20); all samples
apparent for FG10 (Figure 2A). PG10 exhibited were crosslinked with TG (5% w/w) in 1 × PBS.
the longest gelation time of 1169 s (19.4 min) The hydrogels were weighed at the beginning of
(Figure 3C). This measurement was consistent the experiment and every 24 h post-soaking in
with the spreading of PG10 ink at 20 min where 1 × PBS for 4 days. Their swelling ratios were
proper-gelation was observed (Figure 2B). plotted against the incubation time with 1 × PBS.
(Figure 4)
2.5 Effect of preheating on ink viscosity For FG7.5, FG10, PG7.5, and PG10, a decrease

Viscosity is an essential parameter for extrusion- in gel weights was observed post-soaking in 1 ×
based printing as it determines the printability of PBS because 1 × PBS had a much higher ionic
the ink with a given pressure. A previous study has strength than the water contained in each gel. The
shown that the extrusion pressure is proportional to gel placed in the hypertonic environment lost water
the zero-shear viscosity of the extruded filament . due to the osmotic pressure. This effect surpassed
[39]
The time-dependent change in the viscosity of the the ability of hydrogels to absorb water, causing an
[26]
ink required us to control the extrusion pressure overall decrease in the gel weights . FG7.5 and
throughout the printing. We measured the viscosity FG10 showed a 20% reduction in gel weights after
of FG7.5, FG10, and PG10 over time (Figure 3D). 24 h; the gel weights remained relatively constant
The increase in the viscosity of FG and PG was due up till 96 h subsequently. (Figure 4) In contrast,
to the crosslinking of the gelatin by TG. The rate PG7.5 and PG10 exhibited up to 40% reduction
of increase in viscosity was evident by the gradient in gel weights at 72 h, and remained relatively
of the slope reflected in the viscosity-time curves, constant up till 96 h. At a high concentration of
suggesting the required pressure to extrude the gelatin (i.e., FG20 and PG 20), we observed the
ink from the nozzle. The initial sharp increase in swelling of the gel. FG20 showed 5% increase
the viscosity of FG implied that the rapid increase in gel weight after 24 h and remained relatively
in the extrusion pressure was required to ensure constant thereafter until 96 h. PG20 showed a 30%
smooth printing. This rapid increase in the extrusion
pressure resulted in somewhat unpredictable print
quality with discontinuous or spread inks, which was
observed for the FG inks (Figure 3D). The sharp
increase in the viscosity for FG also explains the
short duration of acceptable printability (Figure 2B)
and rapid gelation (Figure 3A, 3B), both of which
made the DIW 3D printing challenging for FG.
In contrast, the increase in the viscosity occurred
slower for PG10 than FG7.5 and FG10 (Figure 3D),
allowing for relatively easy control over the printing
pressure and smooth extrusion of the ink.
2.6 Swelling of hydrogels Figure 4. Swelling of the hydrogels. Changes in
the swelling ratio for the respective FG and TG
The swelling test was performed on the hydrogels over 96 h incubation in 1× PBS. All samples of
prepared from FG and PG. An isotonic environment hydrogels contained 5% (w/w) of TG.

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