Page 565 - IJB-10-6

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

The dry weight loss on day 21 of incubation in [w/w]) within the system proportional to the increment of
PBS is reported in Figure 4D. After seven days, ADA/ Gel (from 0 to 25% [w/w]). In the ADA/Alg/Gel semi-IPN
Alg/Gel_50/50/0 displayed a 30 ± 1% dry weight loss, network, Alg is the main component that interacts with
which remained constant over time. Hydrogels with the calcium ions; hence, the reduction of this component could
lowest Gel content (ADA/Alg/Gel_50/47.5/2.5, ADA/ also lead to decreased hydrogel stability. The presence
Alg/Gel_50/45/5, ADA/Alg/Gel_50/40/10, and ADA/ of Gel in the ADA/Alg/Gel network is also expected to
Alg/Gel_50/35/15) behaved similarly to ADA/Alg/ enhance the enzymatic degradation of the hydrogel, though
Gel_50/50/0, with no statistically significant differences. not tested in this work. Thus, forthcoming investigations
On the contrary, hydrogels with the highest Gel contents should involve in vitro degradation studies in the presence
(ADA/Alg/Gel_50/30/20 and ADA/Alg/Gel_50/25/25) of relevant enzymes (e.g., collagenase).
presented significantly higher dry weight loss compared to
ADA/Alg/Gel_50/50/0 at all the evaluated time points (p 3.3.3. Printability evaluation of Alg/ADA/Gel bioinks
< 0.0021 on days 1, 14, and 21 and p < 0.0001 on day 7 for Based on the rheological and stability properties of ADA/
ADA/Alg/Gel_50/30/20; p < 0.0332 on day 1, p < 0.0002 Alg/Gel samples, three compositions were selected to
on days 7 and 14, and p < 0.0001 on day 21 for ADA/Alg/ be investigated as hydrogel inks for 3D microextrusion
Gel_50/25/25). Moreover, ADA/Alg/Gel_50/30/20 and printing: ADA/Alg/Gel_50/50/0 with no Gel in the
ADA/Alg/Gel_50/25/25 dry weight loss increased over network; ADA/Alg/Gel_50/40/10 having intermediate
time, reaching 53 ± 8% for ADA/Alg/Gel_50/30/20 and 80 Gel concentration; and ADA/Alg/Gel_50/25/25 with
± 6% for ADA/Alg/Gel_50/25/25 after 21 days. the highest Gel content. The three compositions were
selected to evaluate the printability of Alg/ADA/Gel
Biodegradability is a critical requirement in TE bioinks as a platform with tunable degradability, stiffness,
applications, both for regenerative applications and in vitro and cell adhesive properties. Due to the time-dependance
models. Dissolution of ionically crosslinked Alg-based of the internal crosslinking mechanism, printability
hydrogels in the aqueous environment is regulated by the was investigated over time through viscosity tests and
exchange between divalent calcium ions and monovalent microscopy analyses of 3D-printed structures.
cations (e.g., Na ), inducing calcium ion release into
+
the surrounding media. 10,11,37 The dissolution process is Figure 5A–C presents the variations in apparent
usually long-lasting and depends on Alg concentration viscosity over time, studied by shear sweep tests. Regarding
and degree of crosslinking, requiring at least a month to the composition without Gel (ADA/Alg/Gel_50/50/0),
achieve significant initial weight loss. 11,57 The introduction a Newtonian region was detected immediately after
of ADA within the matrix has the advantage of increasing hydrogel preparation (i.e., t = 2 min; time required to load
the dissolution rate due to the higher susceptibility of ADA the sample on the rheometer and start the measurement),
to hydrolytic scission and β-elimination at physiological corresponding to a constant viscosity at low shear rates
pH. 58,79 Moreover, for prospective in vivo applications, ADA (< 1 rad/s). Conversely, a shear-thinning behavior was
with MW lower than 50 kDa (like the one produced in this observed for shear rates higher than 1 rad/s. 80,81 This
work) can be removed from the human body by kidney behavior is typically displayed by non-crosslinked Alg
clearance. 34,57 In our work, ADA/Alg/Gel featured a tunable solutions or hydrogels. 70,80 After 10 min, ADA/Alg/
degradation profile depending on the Gel concentration Gel_50/50/0 displayed a shear-thinning behavior at all
selected, providing a versatile hydrogel platform for TE the shear rates investigated, and this trend remained
applications. The dependence of hydrogel stability on Gel constant over time. The other two hydrogel compositions
content is probably associated with the release of Gel from containing Gel exhibited a shear-thinning behavior for all
the ADA/Alg/Gel semi-IPN network at 37°C (with Gel the time points investigated, without Newtonian behavior
in sol state). Specifically, hydrogels with the highest Gel even immediately after hydrogel preparation.
content (20% and 25% wt.; Table 2) exhibited a dry weight Thereafter, 2D grid structures (15 × 15 mm ) with a
2
loss exceeding the initial Gel percentage, namely 53% and strand spacing of 2.5 mm were then printed at selected
80% wt., respectively, after 21 days of incubation in PBS. time points, and variations in average fiber dimension,
This behavior suggests the release of Gel linked to ADA spreading ratio (S), and printability index (Pr) were
chains (i.e., ADA-modified Gel chains), which were not evaluated as a function of time.
integrated into a broader chemical network. Therefore, the
release of such ADA-modified Gel chains could explain All the compositions displayed a variation in fiber
the higher dry weight loss observed. In addition, decreased diameter as a function of time (Figure 5D–F). ADA/Alg/
stability of ADA/Alg/Gel systems could also be associated Gel_50/50/0 bioinks could be printed at the shortest time
with the reduction of the total Alg content (from 50 to 25% points (i.e., 10 and 20 min), resulting in well-defined 2D

Volume 10 Issue 6 (2024) 557 doi: 10.36922/ijb.4014