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International Journal of Bioprinting 3D-printed hydrogel with antioxidant activity

by a frequency sweep test within the established linear 2.7. Statistical analysis
viscoelastic range at an angular frequency (w of 0.1–100.0 All data were measured in triplicate and were analyzed
rad/s and at 25°C. The G¢ and G² were fitted using Power- using the SAS software (ver. 9.3., SAS Institute Inc., Cary,
Law-type equations, Equations V and VI, respectively. NC, USA). The statistical significance was determined
G¢ = K¢(ω) n¢ (V) using one-way analysis of variance, and mean separation
was calculated using the Duncan’s multiple range test at
G² = K²(ω) ² (VI) p < 0.05.
n
where K¢ and K² are the Power-Law constants, and n¢ and
n² are frequency indices. 3. Results and discussion
The complex modulus (G*) was obtained using 3.1. Morphological, fibrillation, and structural
Equation VII. The gel strength or the intensity of the properties of CMFs during preparation
interaction (A ; Pa·s) of inks and coordination constant (z) Various properties of CMFs produced from high-speed
F
were obtained using the weak gel model (Equation VIII). blending for 0–160 min were investigated (Figures S1 and
S2 in Supplementary File). As blending progressed, long
2
*
G ()ω
ʹ
G ()ω = G ()ω + ʹʹ 2 (VII) and thick intact fibers (diameter of 490–15,000 nm) of
LBKP turned into long but thin fibrils with a diameter of
G* A= F ()ω z / 1 (VIII) 25–54 nm at 80 min and of 16–35 nm at 160 min (Figure S1
in Supplementary File). In addition, the sedimentation
2.6. Characterization of the crosslinked constructs test, which is a measure of the network among microfibrils
The crosslinked construct was further characterized by in cellulose, showed a significant increase in the H /H
27
determining its pH value, antioxidant potential, FTIR value at all concentrations (Figure S2A in Supplementary
s
o
spectra, and texture profile.
File). A longer blending time (160 min) produced more
2.6.1. pH measurement stable CMFs suspensions because of the homogenic,
To determine the pH value, the treated construct (0.1 g) smaller-size fibers/fibrils, as observed in SEM (Figure S1
was dissolved in 5 mL of distilled water, followed by in Supplementary File), which may easily facilitate the
28
homogenization for 2 min. Subsequently, the homogenized formation of networks among fibers/fibrils. This implies
sample underwent centrifugation at 4000 rpm for 20 min, that high flexibility and shear-thinning property of CMFs
after pH were measured three times using a pH meter obtained after longer blending time would cause a better
(Orion Star A211, Thermo Scientific, USA). dispersion of physically entangled network with other
macromolecules, forming a stable composite hydrogel.
2.6.2. Assessment of antioxidant potential of Furthermore, the reduced viscosity of CMFs (i.e., the
crosslinked constructs ratio of viscosity of a dilute polymer solution to net
The antioxidant potential of the borax-treated tested solvent value) increased as the blending time increased
construct was determined using the ABTS radical (Figure S2B in Supplementary File), implying that the
scavenging assay, based on the scavenging of the 2,2’-azino- density of CMFs in the solution increased. This increase
26
bis (3-ethylbenzothiazoline-6-sulfonate) radical anion in density is probably because of the increased specific
(ABTS radical dot+). To generate ABTS radical dot+, a surface area with more numbers of exposed hydroxyl
25
mixture of ABTS stock solution (7.4 mM) and potassium groups after longer blending time. This exposure may
29
persulfate (2.6 mM) in phosphate-buffered saline (PBS, further increase the interactions of CMFs with water
pH 7.4) was prepared and kept in the dark at ambient molecules, as seen in WRV (Figure S2C in Supplementary
temperature for 18 h. A 100 μL of the extracted sample was File). Likewise, the amount of protein bound to cellulose
reacted with 100 μL of ABTS solution in a 96-well plate, was considerably increased as blending progressed, from
and the absorbance was measured at 734 nm. Antioxidant approximately 68.9 μg/g substrate to approximately
activity, expressed as mg/100 g, was obtained by comparing 149.8 μg/g after blending for 160 min (Figure S2C in
the absorbance value to an ascorbic acid standard curve. Supplementary File). Thus, high-speed blending resulted
in continuous extraction, separation, and fibrillations of
2.6.3. Texture profile analysis LBKP, producing CMFs, as reported in a previous study
Texture profiles (hardness, adhesiveness, springiness, and conducting mechanical pretreatment. 21
cohesiveness) of inks were analyzed using a universal
testing machine (Zwick Z010TN, Zwick GmbH & Co.KG, Meanwhile, the FTIR spectrum (Figure S2D in
Ulm, Germany) at 25°C. The speed before and after loading Supplementary File) revealed that the produced CMFs
was set to 30 and 50 mm/min, respectively, and a 1 g pre- showed the typical transmittance patterns of cellulose
load was used. 26 structure, with peaks at 3300 cm (OH stretching and
−1
Volume 10 Issue 1 (2024) 245 https://doi.org/10.36922/ijb.0164