Page 434 - IJB-9-3

International Journal of Bioprinting Gelatin-PVA crosslinked genipin bioinks for skin tissue engineering

at 10 mg for each scaffold. The samples were then boiled 2.11. Rheological characterization
at 100°C for 2 min according to the protocol provided The viscoelastic characteristics of the hydrogels were
by the manufacturer. The amount of free amino groups measured at 23°C using an AR2000 rheometer (TA
was determined using a spectrophotometer (BioTek, Instruments) with a parallel plate geometry (20 mm) and
PowerWave XS, Highland Park, IL, USA) and optical a gap of 2000 µm. The storage modulus (G’), loss modulus
absorbance at 570 nm (Abs570). Besides, different (G’’), and complex viscosity (η*) of the hydrogels were
concentrations of glycine (1.0, 0.5, 0.25, 0.125, and determined using an oscillating mode-frequency sweep at
0.625 mg/mL) were prepared as standard. The crosslinking 23°C with an angular frequency in the range of 0.1 rad/s to
degree was then calculated according to the following 100 rad/s. The viscosity (η) of the bioinks under different
formula: temperatures was evaluated using a flow temperature-ramp

Amino − Amino 1 with a start temperature of 27°C and an end temperature
0
Degree of crosslinking = ×100 of 19°C.
Amino 0
2.12. Porosity degree
Where Amino is the absorbance of non-crosslink
0
hydrogel, and Amino is the absorbance of crosslinked The hydrogels were lyophilized prior to the porosity
1
hydrogel. assessment through a liquid displacement method
that was adapted from Ghaffari et al. (2020) with some
2.10. Antioxidant activity modifications [28] . Absolute ethanol (99.5% EtOH) was
chosen as the displacement liquid due to its capability
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and to penetrate the pores of the hydrogel without causing
2,2’-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS ) shrinkage or swelling of the matrix. The initial weight
+
assays (Sigma, >98% HPLC) were used to determine (W) and the volume (V) of the lyophilized hydrogels
the antioxidant activity of hydrogel samples. Next, the were recorded before being immersed in absolute
hydrogels were then immersed in medium and kept ethanol for 24 h. The excess ethanol was then gradually
at 37°C for 3 days to produce leachate media. In DPPH wiped away using filter paper (Whatmann , No.42,
®
test, a fresh DPPH/ethanol (0.01 µm) solution was used Merck, Darmstadt, Germany), and the weight of the
for the measurements. 5 µL of leachate media were added hydrogel after immersion (W”) was recorded. The
in 195 µL of 100 µM DPPH solution and allow to stand porosity was then calculated using the following
at room temperature for 20 min. The absorbance change equation:
at 515 nm was measured using a spectrophotometer
(" W−
at 734 nm. The DPPH radical scavenging activity was Porosity % () =  W )
calculated using the following formula:   ( × V)   ×100

Abc − Abs Where ρ is the density of the 99.5% EtOH.
Scavengingactivity(%) = ×100
Abc
2.13. Scanning electron microscopy (SEM)
Where Abc is the absorbance of control, and Abs is Field emission scanning electron microscopy (FESEM;
the absorbance of DPPH solution mixed with hydrogel Supra 55VP model, Jena, Germany) was used to examine
samples. Each group had triplicate samples. the cross-sectional microstructure of hydrogels. Before
For the 2,2-azinobis-(3-ethylbenzothiazoline-6- analysis, the lyophilized hydrogels were coated with
sulfonate) ABTS assay, potassium persulfate (K S O ) (104 an ultra-thin layer of gold by ion sputtering. ImageJ
+
2 2
8
mM) was added to ABTS solution in water (1 mL, 7 mM) software (V1.5, Bethesda, MD, USA) was then used
and reacted for 12 – 16 h in the dark to generate an ABTS to randomly measure the average pore sizes of the
+
radical cation solution. The ABTS solution was diluted to hydrogel.
+
a certain concentration with absolute ethanol to obtain a 2.14. Atomic force microscopy (AFM)
working solution with an initial absorbance reading of 0.7
± 0.2. Following that, 10 µL of samples and control were The surface roughness of the lyophilized hydrogel was
added to each well of the 96-well plate. After that, 90 µL characterized using an atomic force microscope (AFM)
of ABTS solution was added into each well and incubated analyzer (Park Systems, NX-10, Korea). The XEI Image
for 4 min in the dark. The scavenging ability was calculated Processing Program was used to analyze the AFM
for each sample according to the absorbance of solvent at photographs, and the roughness of the scaffold surface was
734 nm, and the calculation was similar to what was done assessed. Surface roughness assessment was performed on
to estimate the antioxidant capacity by the DPPH assay. a sample with a sample size 5 × 5 of mm using non-contact

Volume 9 Issue 3 (2023) 426 https://doi.org/10.18063/ijb.677