Page 555 - IJB-10-6

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

membrane tubings were acquired from Spectrum Lab solution (1% [w/v]) was mixed with 25 μL t-BC (30 mM in
(Spectrum Spectra/Por , New Zealand), based on acetic acid) and allowed to react overnight. Subsequently,
TM
TM
regenerated cellulose with MW cut-off (MWCO) of 0.5 mL of TNBS solution (6 mM in 0.1 M sodium
6–8 kDa. Ethanol absolute, anhydrous ethylene glycol bicarbonate) was added and allowed to react for 1 h. The
and hydrochloric acid (37% [w/v]) were obtained from reaction was quenched by adding 0.55 mL of HCl (0.5 N),
Carlo Erba (Italy). Dulbecco’s modified Eagle medium and the absorbance was measured at 340 nm using the
(DMEM), fetal bovine serum (FBS), sodium pyruvate, and UV-Vis spectrophotometer (Varioskan LUX multimode
L-glutamine solution were acquired from GIBCO (USA). microplate, Thermofisher, Italy). The concentration of
CellTiter Blue and CytoTOX-ONE assay were acquired unreacted t-BC was calculated using a calibration curve
TM
from Promega (USA). Live/Dead Viability/Cytotoxicity obtained using t-BC standards and aldehyde groups
TM
Kit was obtained from Invitrogen (USA). determined by calculating the reacted t-BC.
2.2. Production and characterization of 2.2.2. Determination of viscosity average
alginate dialdehyde molecular weight

2.2.1. Production of alginate dialdehyde The viscosity average molecular weight (Mv) of Alg and
Alginate dialdehyde (ADA) was prepared by controlled ADA was determined by viscometric analysis using an
oxidation of sodium Alg via sodium metaperiodate Ubbelohde viscometer (Poulten Selfe & Lee Limited,
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according to the protocol by Sarker et al. Briefly, a 20% England). Briefly, Alg and ADA solutions were prepared
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(w/v) sodium Alg dispersion in ethanol was prepared and at different concentrations (0.025–0.2 and 0.2–0.6%
stirred for 1 h at room temperature. A 0.3 M solution of [w/v], respectively). Inherent and reduced viscosity were
sodium metaperiodate in deionized water was then added calculated and plotted against concentration. Finally,
dropwise to the sodium Alg dispersion under magnetic after checking the linearity, the intrinsic viscosity (η) was
stirring in the dark at room temperature. After 6 h, the derived, and Mv was calculated according to the Mark-
reaction was quenched by adding 0.2% (v/v) ethylene Houwink-Sakurada equation (Equation I). 44
glycol solution under continuous stirring for 30 min.
The resulting solution was dialyzed (Spectrum Spectra/ [η] = K Mv α (I)
TM
Por membrane; MWCO: 6–8 kDa) in deionized water
TM
for seven days to remove all traces of unreacted sodium
metaperiodate and then lyophilized. where K = 0.0073cm /g and α = 0.92 are the Mark-
3
Two methods were used to determine ADA degree Houwink-Sakurada parameters considered for Alg
of oxidation: an indirect and a direct method. For the and ADA. 44
indirect method, the degree of oxidation was determined 2.2.3. Attenuated total reflectance Fourier transform
by measuring the unreacted sodium metaperiodate before infrared spectroscopy
quenching the reaction with ethylene glycol. A soluble Attenuated total reflectance Fourier transform infrared
starch indicator solution was prepared by mixing equal (ATR-FTIR) was carried out to investigate the formation
volumes of KI (20% [w/v]) and soluble starch (1% [w/v]) of aldehyde groups in the Alg chains. The analyses were
solutions using PBS as the solvent. Reaction mixture performed in a spectral range of 4000–600 cm with
−1
(1 mL) was added to 250 mL of deionized water. Then, a resolution of 4 cm using ATR-FTIR Frontier FT-IR
−1
3 mL of the diluted solution was mixed with 1.5 mL of Perkin Elmer instrument (PerkinElmer Inc., USA).
indicator solution and 0.5 mL of distilled water. The
absorbance of the tri-iodine-starch complex was measured 2.2.4. Nuclear magnetic resonance
using a UV-Vis spectrophotometer (Varioskan LUX spectroscopy analysis
multimode microplate, Thermofisher, Italy) at 486 nm. 13 C cross polarization under magic angle spinning
The concentration of periodate in the sample was obtained (CP-MAS) nuclear magnetic resonance spectroscopy
using a calibration curve previously obtained with different (NMR) of Alg powder and ADA lyophilized samples
concentrations of sodium metaperiodate (0.0016–0.02 mg/ was performed using a Bruker AVANCE III HD NMR
mL). The difference between the initial and final amount of spectrometer (Bruker, Germany) equipped with an 11.74 T
sodium metaperiodate corresponds to the hydroxyl groups superconducting magnet (500 MHz H Larmor frequency;
1
converted into aldehyde groups. Bruker, Germany) to investigate the presence of aldehyde
For the direct method, the quantification of aldehyde groups. Moreover, Alg and ADA block composition (M/G
groups was performed using the TNBS assay, following a ratios) was calculated from C CP-MAS NMR spectra
13
protocol described by Wang et al. Briefly, 25 μL of ADA deconvoluted using Gaussian and/or Lorentzian line
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Volume 10 Issue 6 (2024) 547 doi: 10.36922/ijb.4014