Page 483 - IJB-10-6

International Journal of Bioprinting Nanomaterial-bioinks for DLP bioprinting

GelMaGO, and the commercial ink) without cells were 2.9. Porosity of the GelMa-based constructs
measured every day for seven days (n = 6). To measure The density of the GelMa-based constructs (GelMaBB,
the diameter of the construct, an image of the construct GelMaBB-CaP, and GelMaGO) (n = 12) was analyzed
was taken. Using a ruler and the program ImageJ (version using a specific gravity measurement kit (Shimadzu
2.3.0/1.53q), the pixel aspect ratio was calculated. In Corporation, Japan). The solid density in ultra-pure water at
addition, the weight of the constructs was measured with 24°C for cell-free constructs was measured (water density:
a precision scale (BP 211 D; Sartorius Lab Instruments 0.9973 g/cm³). The density ρ was calculated according to
GmbH & Co. KG, Germany) every day for seven days. Equation I. The density of the materials using GelMaBB,
After printing, constructs were directly weighted, which GelMaBB-CaP, and GelMaGO was determined by printing
accounted for day 0. The measurements were performed at solid constructs with the same dimensions as the porous
room temperature. constructs. These solid constructs were measured using the
gravimetry method (n = 4).
2.7. Micro-computed tomography and data analysis
Freeze-dried samples were mounted on custom-built
holders, imaged with a micro-computed tomography (I)
(micro-CT) scanner (VivaCT-80; Scanco Medical AG,
Switzerland), and reconstructed using the manufacturer’s
tomography software (V. 6.4-2). All freeze-dried samples Wa: Solid specimen weight in air, Wl: Solid specimen
were embedded in foam and scanned at a tube voltage of weight in the liquid medium, ρl: Density of the medium
45 kVp, beam current of 177 μA, with a 0.1 mm aluminum liquid. The porosity was calculated with Equation II.
filter, an integration time of 300 ms, a field of view (FOV)
with a diameter of 34.4 mm, 1500 projections/180 °, using (II)
3072 × 3072 sample size, resulting in an isotropic voxel size
of (11.2 μm) .
3
P: Porosity, ρp: Density of the porous construct, ρm
For primary visualization and inspection of the Density of the material (solid construct).
DICOM datasets, the free, open-source software 3D slicer
(https://www.slicer.org/) was used. Histogram-based 2.10. Scanning electron microscopy and surface
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segmentations of the constructs in 3D Slicer were exported structure of printed samples
as 3D meshes (.stl format), which were then cleaned and Cell-free constructs consisting of the different GelMa-
visualized using MeshLab (https://www.meshlab.net). 71 based bioinks (GelMaBB, GelMaBB-CaP, GelMaGO, and
commercial ink) were dehydrated in an increasing alcohol
ImageJ was used to measure the pore sizes by visually series of 50, 60, 70, 80, 90, 95, and 100% ethanol. The final
identifying a region of interest (ROI) along the Z-axis. For step with 100% ethanol was repeated three times to prepare
noise reduction, 3D Gaussian blur filter (1 px) was applied, the samples for critical point drying (LEICA EM CPD
before the maximum intensity projections in the ROIs were 300; Leica Microsystems GmbH, Germany). The samples
created using a limited number of images (GelMaBB = 26 were critical point dried at 40°C and 79–80 bars of CO .
2
images; GelMaGO = 20 images; GelMaBB-CaP = 18 The samples were mounted onto a holder with a carbon
images). The peak positions of line profile plots were used Leit-Tab (Plano GmbH, Germany) and sputter-coated with
to measure the pore wall distances; for GelMaGO and gold-palladium (10 nm thickness) using a Leica EM SCD
GelMaBB-CaP constructs, only pores with small cell wall 500 High-Vacuum sputter-coater (Leica Microsystems
length ratios were used. GmbH, Germany). The samples were scanned at 15 kV
using the scanning electron microscope (SEM) Hitachi
2.8. Water absorption of the GelMa-based constructs TM300 (Hitachi High-Tech, Japan).
The water absorption capacity of the printed GelMaBB,
GelMaBB-CaP, and GelMaGO constructs (n = 12) was 2.11. Live/dead and actin cytoskeleton staining
evaluated. After printing and subsequent washing steps, analyzed by confocal laser scanning microscopy
cell-free constructs were weighed. The samples were then Live/dead staining after 7 and 14 days of culture was
subjected to freeze-drying using a Scanvac Coolsafe 55-9 performed by adding 5 µg/mL calcein AM (Sigma-Aldrich,
PRO freeze dryer (Labogene, Denmark), and the dry USA) to the culture medium to stain living cells; 2 µg/mL
weight was determined. The water absorption capacity was propidium iodide (Sigma-Aldrich, USA) was added to
calculated by the difference between the initial wet weight stain dead cells. The samples were incubated in the staining
and the dry weight. solution for 30 min before washing with a new medium and

Volume 10 Issue 6 (2024) 475 doi: 10.36922/ijb.4015