Page 184 - IJB-9-3

International Journal of Bioprinting Chitin/gelatin/PVA scaffolds

Strain sweeps were carried out between 0.01% and homogeneous cell suspension, after which 96-well and 24-
100% strain at 1 Hz to determine the linear viscoelastic well plates were seeded with a density of 25,000 cells/cm
2
range (LVR) and the critical strain. Then, frequency with 100 and 500 µL of medium, respectively.
sweeps were performed between 0.01 and 50 Hz within the After 24 h of seeding, samples were placed in contact
LVR to obtain elastic modulus (Gʹ) and viscous modulus with the cells by putting them on top of the cells.
(G˝). Finally, the shear flow test was carried out from 0.1 Additionally, some wells were left without biomaterial to
to 50 s . be included as positive and negative controls. For that,
−1
Shear sweep data were fitted to cross model for shear the samples had been previously cut in 5 mm and 8 mm
thinning fluids , represented in Equation IV: diameter discs for 96-well and 24-well plates, respectively,
[29]
washed by dialysis in Dulbecco’s PBS (DPBS) (Gibco) for
η − η
η η + 0 ∞ m (IV) 72 h, and sterilized by immersion into 70% ethanol for
=
∞
1 + C  γ ( ) 5 min and exposing to UV for 15 min. Finally, samples
where C is consistency or cross time constant, γ˙ shear rate, were washed three times for 5 min with DPBS in sterile
η viscosity, η , and η viscosity at low and high shear values conditions before placing them into the wells.
0
∞
(zero and infinite shear values), and m dimensionless cross Short-term biocompatibility was evaluated in 96-well
rate constant, calculated by curve fitting in the slope region. plates at 24, 48, and 72 h of cell culture after the exposure
of the scaffolds. Cell activity was measured based on the
2.3.9. Mucoadhesion test reductive capacity of the cells using the colorimetric assay
Mucoadhesion test was carried out using a TA.XT.Plus C Cell Counting Kit-8 (Sigma). Following the manufacturer’s
Texture Analyzer (Aname, Madrid, Spain) with a 5 kg load recommendations, the wells were first washed with
cell. Data were collected using 0.1 mm/s test speed, 30 s DPBS and then, the compound was added to the wells at
contact time, 3.2 g trigger force, and 20.4 g applied force. a final concentration of 1:10 in cell medium. After 2 h of
The biological substrate used was a filter paper moistened incubation at 37°C, the absorbance was measured at 450 nm
during 3 min with 1% type II mucin from porcine stomach in the Halo Led 96. Cell mortality was assessed according
(Sigma-Aldrich, Madrid, Spain). to plasma membrane integrity; for that, the Cell-Tox Green

2.3.10. Texture profile analysis Cytotoxicity Assay (Promega) was used. To this end, the
A TA.XT.Plus C Texture Analyzer (Aname, Madrid, Spain) protocol recommended by the manufacturer was followed:
with a 5 kg load cell was used to obtain texture profile the dye was prepared at a concentration of 1:500 in the given
analysis (TPA). Data were collected at 1 mm/s speed, 20% buffer and 100 µL were directly added to the wells to a final
strain, and 5 g trigger force. concentration of 1:1000. After 15 min of incubation at 37°C
and protected from light, the fluorescence was measured in
2.3.11. Optical microscopy the Promega GloMax Discover at a wavelength of 492 nm
Surface characterization was carried out using Nikon excitation and 535 nm emission.
Eclipse E600 optical microscope with digital camera (Izasa Long-term observation was performed at 24 h, 72 h,
Scientific,) equipped with 10× objective and 10× eyepieces. and 7 days after the placement of the scaffold through the
Images were analyzed with Image J software . Live/Dead Cell Viability Assay (Thermo Fisher) in order
[30]
to monitor directly the cell status and phenotype. For
2.3.12. Biocompatibility assessment that purpose, calcein AM and ethidium homodimer-1
The biocompatibility assessment was performed in were added to a final concentration of 1:2000 and 1:1000,
compliance with the requirements of ISO 10993-5 . respectively, from which 200 µL of the preparation were
[31]
Human fibroblast HS27 (ECACC) cell line was used for the added to the 24 wells. After 15 min of incubation, images
biocompatibility determination. Following the manufacturer were taken with the ZEISS LSM 900 confocal microscope.
recommendations, fibroblasts were cultured on Dulbecco’s
Modified Eagle’s Medium (DMEM) (Sigma), supplemented In addition to the study conditions, positive (CTR+)
with 10% (v/v) inactive fetal bovine serum (FBS) (Lonza), and negative (CTR-) controls were included. For
1% (v/v) penicillin-streptomycin (Lonza), and 1% (v/v) CTR+, cells seeded in the same conditions, but without
L-glutamine (Sigma) at 37°C in a humidified incubator biomaterial, were used, while cells were treated with lysis
with a 5% CO atmosphere. Cell passages were performed solutions to provoke cell death for CTR-. In the case of the
2
weekly until confluence. At that point, cells were collected Cell-Tox Green Cytotoxicity Assay, the given buffer was
by 0.25% trypsin-EDTA (Sigma) and centrifuged at 1500 employed and, in the case of CCK-8 and Live/Dead Cell
rpm for 5 min at room temperature. The obtained pellet was Viability Assay, dimethyl sulfoxide (DMSO) (Sigma) was
resuspended in the above-mentioned medium to obtain a used instead. Each condition was analyzed in fourfold.

Volume 9 Issue 3 (2023) 176 https://doi.org/10.18063/ijb.701