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Tan, et al.
chemical crosslinking, the formation of a rigid cells preferentially grow and differentiate on soft,
network reduces the extent of swelling in the tissue-like substrates [33-35] . We also included FG10
resulting hydrogel . In this study, we investigated for our later study as a control for preheating;
[26]
the effect of swelling of FG and PG crosslinked FG10 and PG10 contained the same gelatin
with TG. concentration, and the observed differences, if
any, shall be attributed to the consequences of
2.2 Evaluation of hydrogel stiffness preheating. After identifying suitable inks (that is,

Initially, stiffness of hydrogels prepared from FG7.5, FG10, and PG10), we proceeded to assess
FG and PG crosslinked with TG (5% w/w) was their printability.
investigated. Stiffness is an essential mechano- 2.3 3D printing of gelatin and TG inks and
physical property that influences cell growth and printability characterization
function . We intended to show that PG would
[27]
be able to confer better printability than FG for The printability of the inks was assessed at selected
the hydrogels yielding similar stiffness. We have time points using the printability value (Pr) . Pr
[36]
chosen hydrogels yielding an estimated elastic indicated the degree of gelation of the extruded
modulus of 10 kPa, which is tissue-like stiffness filament at the respective time points . Three states
[36]
deemed suitable for the culture of many cell types, of gelation were considered for the printed inks:
including myoblasts, human primary keratinocytes, Under-gelation, proper-gelation, and over-gelation.
and human embryonic stem cells [28-30] . Amplitude Under-gelation would yield obvious chamfers in
sweep tests were conducted for FG and PG with the printed grids due to the fusion of the subsequent
the concentrations ranging from 5 to 20% w/w two layers of the interconnected filaments (Pr < 1).
while keeping TG concentration at 5% w/w. We During proper-gelation, the interconnected filaments
use the following nomenclature to describe the would demonstrate a perfect square shape or close to
composition of FG and PG throughout the current a square with regular edges (Pr = 1). Over-gelation,
study. For instance, FG10 indicates that the ink however, led to the formation of irregularly printed
contained 10% w/w FG and 5% w/w TG, while and interconnected filaments (Pr > 1). The larger
PG10 indicates that the ink contained 10% w/w values of Pr indicated the higher degree of gelation
PG and 5% w/w TG. The hydrogel stiffness was while the smaller Pr indicated the lower degree of
determined by its elastic modulus, which was gelation. We set the acceptable range of Pr to be
estimated from the storage modulus obtained 0.9 – 1.1, as reported previously with 3D printed
from the viscoelastic region of the flow diagrams, hydrogel constructs .
[36]
as reported. The estimated elastic modulus of FG7.5, FG10, and PG10 were printed in a grid
[31]
both FG and PG hydrogels increased with gelatin pattern on a glass surface at 3 min, 5 min, 10 min,
concentrations. Exposure of gelatin to high and every 5 min after that. The printing was
temperatures for extended periods resulted in stopped when the inks became clumpy or when
the thermal hydrolysis of the polypeptide chains. the ink could not be extruded at the maximum
Thermal hydrolysis caused a decrease in the gel pressure of the dispenser. The Hagen-Poiseuille
rigidity (i.e., gel strength) . The stiffness of equation suggested that the volumetric flow rate
[32]
hydrogels of FG7.5 was similar to PG10, both was inversely proportional to the viscosity of
of which yielded an estimated elastic modulus of fluids . Continuous increase in the viscosity
[37]
approximately 10 kPa ( Figure 1). We chose these required compensation by the increase in the
two hydrogel compositions for our subsequent applied pressure. We observed that extruding FG
work because they yielded a tissue-like stiffness inks were more challenging than PG due to the
which deemed suitable for the culture of many cell rapid increase in the viscosity (Figure 2A and 2B).
types. Highly stiff substrates were not favored for Under-gelation was observed for FG7.5 at 3
cell cultures, as multiple studies have shown that min but not for FG10 (Figure 2A). The duration

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