Page 260 - IJB-10-2

International Journal of Bioprinting 3D bioprinting of boluses for radiotherapy

powders. Subsequently, PA, PEGDA (0.2 mol% of AM), with height and diameter measuring 10 mm and 18 mm,
LAP (0.1 wt%), and tartrazine (Tar; 0.0035 wt%) were respectively. The compression speed was set at 5 mm/min.
gradually added under stirring to create the gel precursors. To ensure accuracy and repeatability, all samples were
Subsequent procedures were carried out in the absence tested in triplicate under consistent conditions.
of light due to the high photosensitivity of the initiator. The elastic modulus of the gels was determined by
A series of water-to-glycerol ratios, i.e., 50%, 70%, and calculating the stress–strain curve within a strain range
80%, were tested to investigate their impact on the bioink, of 5% to 10%. Additionally, tensile-adhesion tests were
while different concentrations of ALG (0.5 wt%, 1 wt%,
and 2 wt%) were examined to assess their influence on the cyclically performed on porcine skin to evaluate the
mechanical properties of the resulting gel. Additionally, repeatability of the gel’s adhesive properties. Adhesion
three distinct concentrations of PA (0.5 wt%, 3 wt%, strength was calculated as the maximum force divided by
and 6 wt%) were prepared to evaluate its anti-bacterial bonded area.
and adhesive properties. All bioinks were stored away 2.4. Biological properties
from light. 2.4.1. Evaluation of anti-bacterial efficacy
To investigate the effect of different components on To assess the anti-bacterial efficacy of the specimens
the properties of the hydrogel, five hydrogel samples were against Staphylococcus aureus (ATCC6538), quantitative
fabricated and labeled as PAM (polyacrylamide), PAM/ evaluations were performed using both Live/Dead staining
ALG, PAM/ALG/PA, PAM/ALG/PA (GW), and PAM/ assay and spread plate analysis. The bacteria were cultured
ALG/PA/Tar (GW), in which GW denotes that the hydrogel in a sterile liquid medium consisting of 1 g peptone, 0.5 g
was formed in glycerol-water (GW) binary solvent. beef extract, and 0.5 g NaCl in 100 mL deionized water.
A bacterial suspension of 1 × 10 CFU mL was then
-1
6
2.3. Physicochemical properties incubated with the gels in a 24-well plate. Subsequently,
2.3.1. Structural characterization the treated bacterial suspension was diluted to 1 × 10 CFU
3
To assess the impact of ALG and PA on gel microstructure, mL , then the diluted bacterial suspension (80 µL) was
-1
composite gels were subjected to freeze-drying and spread onto agar medium and incubated for 24 h, followed
subsequently coated with gold/palladium. The cross- by colony counting on the agar plate to determine anti-
sectional morphology of the gels was examined using a bacterial efficacy. The tests were conducted in triplicate to
scanning electron microscope (SEM; JSM-7001F, JEOL, ensure reliability.
Japan). To investigate the structure of the composite gel,
Fourier-transform infrared spectroscopy (FTIR) spectra 2.4.2. Cell viability and proliferation
(Bruker Daltonics, Bremen, Germany) were obtained at NIH-3T3 cells (mouse embryonic fibroblast, ATCC CRL-
-1
22°C over a range of 500 to 4000 cm . 1658) were employed to evaluate the cytocompatibility
of the composite gel. Initially, the gels were prepared in
2.3.2. Photoresponsive performance 24-well plate and subsequently diluted gel leaching were co-
The photocuring time of the bioink was determined using cultured with cells (at a density of 2 × 10 cells per well) for 1,
4
a rheometer (MCR 702 Anton Paar) equipped with an 3, and 5 days. Fifty microliter of a solution containing live/
optics attachment (405 nm). The bioink was loaded onto dead fluorescent dyes (0.5 µL mL calcein AM and 2 µL
-1
a parallel plate with a gap size of 200 μm. An ultraviolet mL ethylenediamine homodimer (EthD-1)) was added
-1
2
light with an intensity of 2 mW/cm was used to illuminate to the slides, followed by a 40-min staining process in
the plate window, and the time required for photocuring the dark. Following washing with phosphate-buffered
was measured.
saline, confocal laser scanning microscopy (CLSM) was
Additionally, a rheometer was employed to quantify the utilized to capture images of green viable cells and red
viscosity of the solution. The gel’s viscosity was assessed at non-viable cells.
ambient temperature by rotating it at a rate of 2 revolutions Furthermore, the cytocompatibility of the composite
per second using a 25 mm parallel plate clamp. Moreover,
a sweep test was conducted with an angular frequency of gels was assessed by means of the 3-(4,5-dimethylthiazol-
6 rad/s to monitor the temporal evolution of both loss 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The
modulus and storage modulus for gels. experiments were carried out in triplicate. The process
entails inoculating 1 mL of cell suspension containing 2 ×
4
2.3.3. Mechanical properties 10 cells onto a well of 24-well plate, followed by addition
The mechanical properties of the gels were evaluated of different gels. After co-culturing for 1, 3, and 5 days,
using a universal testing machine (RGM-2010, Reger, a mixture of 900 µL Dulbecco’s Modified Eagle Medium
China). Tensile tests were performed on the gel samples, (DMEM) and 100 µL of a 5 mg/mL MTT solution was

Volume 10 Issue 2 (2024) 252 doi: 10.36922/ijb.1589