Page 187 - IJB-10-1

International Journal of Bioprinting Nanoclay biopolymer inks for 3D printing

2.3. 3D printing of hydrogel inks The degradation behavior of each sample was evaluated
The hydrogel-based inks were printed using the 3D by immersing the specimens in water or PBS. The
Bioprinter 3D Discovery TM (RegenHU Ltd., Switzerland, hydrogels were kept at 37°C for up to 7 days. The weight
Villaz-St-Pierre). The printing process was conducted of equilibrium swollen samples was considered the initial
using a direct dispensing print-head and a 5-mL syringe weight. The hydrogel degradation degree was computed
with an attached cylindrical nozzle, under varying printing as the weight drop (w–w0) relative to the weight of the
pressures and speeds at room temperature. The generated equilibrium swollen samples (w0). The measurements
3D structures were crosslinked following the printing were performed in triplicate.
process by their submersion in a 2%wt. CaCl solution
2
for an hour. Crosslinked 3D structures were then washed 2.6. Morphological and structural analyses
thoroughly with distilled water and were subsequently Fourier transform infrared (FTIR) spectroscopy was
freeze-dried. Acquired dried samples were stored in a used to structurally characterize the samples containing
desiccator at room temperature. alginate, salecan, and nanoclay. The analyses were carried
out using a Bruker Vertex 70 FTIR spectrometer (Bruker,
2.4. Determinations of gel fraction and the Billerica, MA, USA). FTIR spectra in the 4000–400 cm
-1
evaluation of salecan stability for the alginate– wavenumber region were captured.
salecan-based hydrogel samples Environmental scanning electron microscopy
The determination of biopolymers gel fraction was (ESEM-FEI Quanta 200, Eindhoven, The Netherlands)
performed as follows: each 3D-printed sample was weighed was employed to analyze the morphology of the printed
and was introduced in 20 mL of deionized water for 24 h lyophilized samples and their 3D structures, without being
in distilled water at 40°C. In this way, all the uncrosslinked sputter-coated.
biopolymers were dissolved, and we were able to determine
the sol part. The extracted hydrogel samples were freeze- Computer microtomography analysis was performed
dried and weighed again. Equation I was used to measure using Bruker µCT 1272 high-resolution equipment. The
the samples gel fractions: samples were fixed on a support and scanned during a 180°
rotation, with an image pixel size of 5 µm (one pixel depicting
% Gel fraction= Mf × 100/Mi (I)
5 × 5 µm from the physical sample), at 70 kV, 130 µA, 500 ms
where Mf = final weight of the freeze-dried construct and exposure time and at a rotation step of 0.25°. Scanning was
Mi = initial weight of the construct. performed without filter, and each 2D projection was the
The stability of salecan chains entangled in alginate average of five consecutive frames. Each dataset contained
networks was evaluated by using the phenol sulfuric acid 1080 2D projections (2 × 1640 pixels) which were further
method. 36,39 Thus, the content of salecan in the washing used in NRecon software to generate the 3D tomograms.
solutions resulted from the extraction process and was All quantitative measurements were performed in CTAn
evaluated by UV-VIS measurements, as previously software, on the reconstructed tomograms.
described. The amount of salecan which remained well X-ray diffractometer (Rigaku Ultima IV, Tokyo, Japan)
39
entangled in the alginate networks was then determined. was used to ascertain the structure of the nanocomposite
For the samples including inorganic partner, only samples. At 40 kV and 30 mA, Cu Kα radiation (λ=1.5406 Å)
the biopolymeric content was taken into account in was employed. Under air pressure and room temperature,
calculating the gel fraction as well as for the evaluation of all of the analyses were conducted on samples in powder
interpenetrated salecan. form. The scanning speed was 1°/min, and the data
collection interval was 2θ range 1–50°.
2.5. Swelling and degradation analyses
The hydrogel-based 3D-printed dry samples were weighed 2.7. Rheological and mechanical analyses
and submerged in solutions with different pH (pH = 2, A Kinexus Pro rheometer (Malvern) with plate–plate
5.5, and 7.4). Readings of the swollen samples weight were geometry (upper plate diameter = 20 mm) was employed
taken at each hydrogel’s equilibrium time. This experiment to monitor the flow behavior of the synthesized materials.
was performed in duplicate. Swelling degree was calculated The gap between the two plates was maintained constant at
using Equation II: 0.5 mm throughout the testing. The apparatus was equipped
%ESD = (m equilibrium swollen sample - m dry sample ) × 100/m dry sample with Peltier element for precise temperature control and a
(II) stainless-steel hood to prevent dehydration during testing.
To obtain information regarding the processability of the
where ESD is equilibrium swelling degree, and m is the formulations, flow curves were registered in the shear rate
weight of the sample in each case. interval of 10 to 10 at a working temperature of 25°C.
3
-3
Volume 10 Issue 1 (2024) 179 https://doi.org/10.36922/ijb.0967