Page 560 - IJB-10-6

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

reported contradictory results on the effect of oxidation on exhibited higher G′ than G″, demonstrating their hydrogel
G and M groups, suggesting either a preferential reaction of state. G′ increased as a function of both Alg content and
G groups to oxidation 52,53 or no difference between the two CaCO concentration (Table S2, Supporting Information).
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groups. Differences in the results might be associated with At 3% (w/v) CaCO , samples with the highest Alg content
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the different M/G ratios of the starting Alg being oxidized (ADA/Alg_50/50_C3) exhibited higher G′ compared to
and other factors, such as M and G block distribution. ADA/Alg_66/33_C3 (p < 0.0002). Indeed, Alg is primarily
Finally, ADA Mv was calculated to be 22 ± 1 kDa based responsible for the internal crosslinking mechanism of
on the Mark-Houwink-Sakurada equation, corresponding ADA/Alg hydrogels. ADA has a reduced ability to undergo
to a 93.5% reduction relative to Alg Mv (340 ± 5 kDa). crosslinking with calcium ions due to the chemical
50,51,57
Oxidation by sodium periodate triggers the scission of modification of its chains and reduced MW. Ring-
polysaccharide chains, causing a decrease in ADA MW and opening of ADA chains upon oxidation weakens or even
intermolecular interactions. Coherently to the literature, hinders ionic crosslinking, which requires the presence
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MW reduction between 80 and 90% was thus expected of approximately 20 consecutive guluronate groups for
34,58
with an oxidation degree of 25–30%. 51,56 forming egg-box ionic junctions with calcium ions.
In this work, an initial characterization of ADA (data
3.2. ADA/Alg-based hydrogels not shown) confirmed its low ionic crosslinking ability
The ADA/Alg samples were first optimized by varying the in the presence of CaCO and GDL, as suggested by the
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Alg:ADA ratio, as well as CaCO and GDL concentrations, formation of soft hydrogels with low G′ (< 100 Pa) even at
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to obtain suitable stiffness for potential cardiac TE high polymer concentrations (i.e., 10% [w/v]).
applications. Hydrogels with the compositions reported in At the same Alg:ADA ratio, hydrogels with higher calcium
Table 1 were characterized through frequency sweep tests. content (i.e., ADA/Alg_50/50_C6) exhibited higher
Initially, the influence of the Alg:ADA ratio and calcium G′ compared to those with lower CaCO content (i.e.,
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concentration was studied at fixed GDL content (Figure 2A ADA/Alg_50/50_C3) (p < 0.0001). Overall, the ADA/
and B). As displayed in Figure 2A, all compositions Alg_50/50_C6 composition was selected as it displayed the

Figure 2. Rheological analysis of Alg/ADA hydrogels. (A) Storage modulus (G′; continuous line) and loss modulus (G″; dotted line) of ADA/Alg_50/50_
C6 (red), ADA/Alg_50/50_C3 (orange), ADA/Alg_66/33_C6 (yellow), ADA/Alg_66/33_C3 (light blue), and ADA/Alg_66/33_C1.5 (blue) as a function

of angular frequency (0.1 and 100 rad/s) at 37°C (n = 3). (B) Elastic modulus (E) of ADA/Alg_50/50_C6 (red), ADA/Alg_50/50_C3 (orange), ADA/

Alg_66/33_C6 (yellow), ADA/Alg_66/33_C3 (light blue), and ADA/Alg_66/33_C1.5 (blue), derived from frequency sweep tests (n = 3). **** p < 0.0001

indicates a statistically significant difference with ADA/Alg_50/50_C6. Abbreviations: Alg: Alginate; ADA: Alginate dialdehyde.
Volume 10 Issue 6 (2024) 552 doi: 10.36922/ijb.4014