Page 330 - IJB-10-4

International Journal of Bioprinting 3D bioprinting of composite hydrogels

G-code. The cube printing pattern (10 × 10 mm) was five W
layers thick with a 25% infill density, rectilinear internal Gel fraction (%) = W 2 × 100 (I)
fill pattern, and internal and external infill angle offsets of 1
90°, −90° and 45°, −45°, respectively. The corneal model
(12 × 12 × 3 mm) had an extrusion width of 0.413 mm 2.4. Transparency
and an outline overlap of 50%. All samples were printed Transparency of the hydrogel samples was measured by
using a 22G needle at 300 mm/min and a pressure detecting the percentage of light transmittance. For this,
of 1 bar at room temperature. For support structure the 3D-bioprinted hydrogel scaffolds (n = 4) were punched
generation, a corneal model designed in SolidWorks into thin discs of 6 mm in diameter and transferred to a
2023 with dimensions of 19.97 mm in length, 20 mm 96-well plate. Next, 100 μL of deionized water was added to
in width, and 3.5 mm in height was made of glass and all wells. Pure water without a bioprinted sample was used
used as a template for 3D bioprinting. A pneumatic N2- as a control. The absorbance was read using a Biochrom
3Dbioprinter (3DPL Corporation, USA) was employed to Biowave2 spectrophotometer (Biochrom, UK) in the range
print the models. The support was fixed on the bioprinter of 300–700 nm. The percentage of light transmittance (%T)
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bed during the bioprinting process. Bioprinting of the was calculated according to Equation II.
corneal model began from the center of the glass support
and extended toward the rim. After bioprinting, the 3D T % () = 10 (− absorbance) ×100 (II)
hydrogels were freeze-thawed four times and then heated
at 37°C overnight until completely dry.

2.3. Hydrogel scaffold characterization 2.5. In vitro degradation
The morphology of the bioprinted hydrogel samples To investigate the stability of 3D-bioprinted scaffolds,
was observed by optical microscopy (CKX53; Olympus, in vitro degradation was conducted. The pre-weighed
Japan). To investigate interactions between GG and scaffolds (m ) (n = 3) were immersed in phosphate buffer
1
PEI, Fourier-transform infrared (FTIR) spectroscopy saline (PBS, pH 7.4; Sigma-Aldrich, USA) and then
was carried out using a Bruker Vector33 spectrometer placed in an incubator at 37°C for 3, 7, and 14 days. After
(Bruker, USA) in the range of 400–4000 cm . The contact these times, the samples were washed with deionized
−1
angle was evaluated using a CA-500A contact angle water to remove excess PBS, dried, and reweighed
measurement system (Iranlabexpo Co., Iran) at room (m ). The degradation rate was measured according to
2
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temperature at three points on the surface of each sample. Equation III.
Zeta potential analysis was performed using a Zetasizer
(3000HSA; Unigreenscheme Co., UK) with samples m − m 2
1
soaked in deionized water. To measure free amine groups Degradation rate (%) = m × 100 (III)
in the structure of the hydrogel, the amine-functionalized 1
bioink was dissolved in deionized water and then added
to 12 mM ninhydrin solution (Merck, USA) prepared 2.6. Mechanical properties
in ethanol (Merck, USA) in a 1:1 ratio of ninhydrin to To evaluate the structural integrity of the samples, the
sample. The sample was then placed in a boiling water mechanical strength was measured. The tensile strength
bath for 15 min and then cooled at room temperature for was quantified in both wet and dry conditions using an
10 min. The absorbance of the solution was measured at stm20 testing machine (Santam Co., Iran) equipped with
560 nm using a Biochrom Biowave2 spectrophotometer a crosshead with a load cell of 10 N and a crosshead speed
(Biochrom, UK). Pure PEI was used to obtain a standard of 5 mm/min. The specimens (n = 4) were molded in the
curve by measuring the absorbance of PEI at different form of rectangular strips with dimensions of 20 × 10 × 0.5
concentrations. mm. For measurement in the wet state, before each test,
the specimens were soaked in PBS for 1 h at 37°C.
A gel fraction test was performed to assess the gelation
of the bioink. For this purpose, a film with approximately 2.7. Rheology
the same dimensions as the printed structure was prepared, The rheological behavior of the bioinks was assessed
dried at 60°C overnight, and then weighed (W ). The film through the flow curves of each bioink using a Physica
1
was then immersed in deionized water at room temperature MCR301 rheometer (Anton Paar, Austria) equipped with
for 3 h to remove any ungelled parts. The immersed films a plate-plate geometry diameter (D = 25 mm). Shear-
were again dried at 60°C overnight and weighed (W ). The thinning behavior was observed in flow mode by varying
2
gel fraction was calculated according to Equation I. the shear rate from 1 to 100 s for 60 s at 37°C.
−1
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Volume 10 Issue 4 (2024) 322 doi: 10.36922/ijb.3440