Page 331 - IJB-10-4

International Journal of Bioprinting 3D bioprinting of composite hydrogels

2.8. Drug loading and release 2.10. Statistical analysis
For drug loading, bioinks were prepared according to Data analysis was conducted in Excel 2016. Statistically
Section 2.1. with 0.1% (w/v) BSP (Sina Darou Co., Iran), significant differences between mean values were
as suggested in previous studies, 70,71 added to GG–PEI calculated using either a t-test, one-way analysis of variance
solution under magnetic stirring at 30°C for 30 min (BSP– (ANOVA), or two-way ANOVA, with p < 0.05 indicating
GG–PEI). Loading efficiency was measured by immersing significance. All data are presented as mean ± standard
the hydrogels in 5 mL PBS contained in 15 mL conical error of the mean.
tubes. The samples were incubated at 37°C with shaking.
The release of BSP was measured at different time intervals 3. Results and discussion
(5, 15, 30, and 45 min; 1, 1.5, 2, 3, 4, 5, 6, 7, 8, and 24 h) by
ultraviolet (UV) spectrophotometry (Biochrom Biowave2; 3.1. Bioink characterization
Biochrom, UK) at a wavelength of 246 nm. The amount A glass support was used to facilitate the printing of
of released BSP was determined by interpolating from a a corneal structure and maintain the shape of printed
calibration curve obtained for known concentrations of BSP. corneal constructs during and after printing (Figure 1a).
An original model was designed in SolidWorks to export
2.9. Cell behavior G-code for printing the corneal structure (Figure 1b).
L929-RFP mouse corneal fibroblasts (Merc, Iran) were The GG–3PEI composite corneal scaffold displayed a
cultured in Dulbecco’s Modified Eagle Medium/Nutrient high degree of transparency as the GG transparency was
Mixture F-12 (DMEM-F12), supplemented with 10% maintained after blending with 3% PEI (Figure 1c–e).
fetal bovine serum (FBS) and 1% penicillin/streptomycin Additionally, porous cube-like structures were printed to
(Merc, Iran). The cells were maintained in T-25 cell monitor the shape fidelity and printability of the various
culture flasks in a 5% CO incubator at 37°C. The cells bioink formulations. The surface morphologies of the
2
were passaged at 85% confluency. Cell suspensions were bioprinted hydrogels demonstrated that the GG and GG–
obtained following trypsinization. Prior to cell culture, 3PEI composite hydrogels retained their grid structures
all bioink/hydrogel samples were sterilized under UVC in a repeating well-ordered pattern (Figure 1f and g). The
radiation for 30 min. Thereafter, the samples were placed GG–3PEI strands and the edge of square pores were thicker
in a 96-well culture plate, seeded at 1 × 10 cells/well, and rounder compared to GG due to the lower viscosity
6
and incubated under 5% CO at 37°C. After 24 and 72 and reduced gelation of the GG–-3PEI bioink. However,
2
h of incubation, the medium was removed and replaced both formulations demonstrated good shape fidelity and
with resazurin (1% [v/v]) for 4 h (37°C, 5% CO ). Cell structural integrity. The bioprinted GG–3PEI composite
2
viability was determined by measuring absorbance at hydrogel was transparent as assessed qualitatively by the
570 nm using a spectrophotometer (Biochrom Biowave2; ability to clearly view the text through the material in air
Biochrom, UK). Each test was performed in triplicate. and when immersed in PBS (Figure 1h).
Viability was further confirmed after 5 days of cell Fourier-transform infrared (FTIR) spectroscopy was
culture by acridine orange (AO) (green) staining. used to investigate molecular interactions between GG
The AO stain (Merc, Iran) was applied for 10 min and PEI. As represented in Figure 2a, for pure GG, sharp
before observation using an inverted fluorescence absorption bands were observed at 3435, 2922, 1625, and
microscope (CKX53; Olympus, Japan). 1422 cm , corresponding to stretching vibrations of O–H,
−1
Cell attachment and morphology were evaluated C–H, and asymmetric and symmetric C=O (characteristic
by scanning electron microscopy (SEM, S360- for carboxyl), and the C–C vibration mode of GG,
−1
Cambridge; Cambridge, UK). For this, the cell respectively. The peaks at 598–1010 cm are attributed to
6
suspension (1 × 10 cells/well) was seeded on the C–O–C symmetric stretching in the GG structure. For PEI,
−1
surface of the hydrogels contained in 24-well cell the characteristic peaks at 3455 and 1646 cm are related
20
culture plates and incubated at 37°C under 5% CO . to N–H stretching and bending vibrations, respectively.
2
−1
After 5 days of cell culture, the samples were washed The bands at 2076 and 724 cm are due to the vibrations
with PBS and fixed with 2.5% (w/v) glutaraldehyde of C–H and C–N, respectively. In the GG–PEI composite
(Sigma-Aldrich, USA) for 2 h. The samples were scaffold, the peaks for asymmetric and symmetric
then dehydrated in a series of ethanol solutions (60%, carboxylate groups shifted to 1595 and 1415 cm , and a
−1
−1
70%, 80%, 90%, and 100%) (Sigma-Aldrich, USA) new specific peak appeared at 1715 cm , indicating an
for 5 min in each solution and finally transferred to electrostatic interaction between the amine groups of PEI
100% ethanol for 15 min. and the anionic carboxylate groups of CA that was used as

Volume 10 Issue 4 (2024) 323 doi: 10.36922/ijb.3440