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International Journal of Bioprinting Fine collagen scaffold for osteogenesis

printing (inner diameter: 200 μm; outer diameter: 220 μm). 2.8. Cell culture and characterizations
The temperature of the 3D cryogenic well was set to −1°С
for printing both collagen I and gelatin-based inks, and 2.8.1. Cell seeding
a total of seven layers were printed with each ink. Three MSCs were extracted from 2-week-old Sprague Dawley
samples were printed using ink B and with rod distances mice. Before seeding cells, all scaffolds (samples 1–4) were
(distance between the centerline of adjacent rods) of immersed in 75% ethanol for 10 min, washed once with
300, 450, and 600 μm, and were denoted as “sample 1,” phosphate-buffered saline solution (Beyotime, China),
“sample 2,” and “sample 3”, respectively. Another scaffold exposed to UV radiation for 30 min, and immersed in an
(α-MEM) Minimum Essential Medium (Vivacell, China)
was printed with gelatin-based ink and a rod distance containing 10% fetal bovine serum (Gibco, Australia),
of 600 μm, denoted as “sample 4”. The layer height and penicillin (10 kU/mL) and 10 mg/mL streptomycin
extrusion pressure were both set to 0.14 mm. The printing solution (10 mg/mL, Beyotime, China) for 24 h. This
pressure was set to 0.18 MPa for printing collagen I-based modified medium was denoted as “complete medium”.
ink and 0.25 MPa for printing gelatin-based ink. Upon MSCs (passage 2) were seeded onto the scaffolds at a
the completion of printing, the crosslinking solution was density of 10 cells/mL. Cell culture was conducted in an
6
added dropwise to the scaffolds until fully immersed. incubator (Thermo Fisher Scientific, USA) at 37°С with
The scaffolds were then removed and stored in a refrigerator 5% CO . After 24 h, once cell adhesion was observed,
2
at −20°С. the complete medium was replaced with an osteogenic
2.4. Morphological study induction medium containing an additional 50 μM
All printed scaffolds were freeze-dried and loaded into the ascorbic acid, 10 mM β-glycerol phosphate, and 100 nM
chamber of a scanning electron microscope (SEM; Zeiss, dexamethasone (all from Sigma Aldrich, USA).
Germany) and vacuumed again. SEM observation was 2.8.2. Cell counting kit-8 test
conducted under a secondary electron (SE) mode with On days 1, 3, and 5 of culture, a cell counting kit (CCK)-
an accelerating voltage of 3kV. ImageJ software (1.8.0, 8 test was conducted to assess cell proliferation. The
National Institute of Health, USA) was used to analyze the α-MEM medium containing 10% CCK-8 cell counting
average rod diameter and pore size of all scaffolds. agent (Beyotime, China) was added and incubated for 1 h.
A total of 100 μL of cell supernatant was used to measure
2.5. Rheology the Optical Density (OD) value at a wavelength of 450 nm
Rheological studies were conducted with an Anton Paar using a spectrophotometer (Multiskan GO, Thermofisher
MCR 302e rheometer (Anton Paar, Austria), equipped with Scientific, USA).
a 25 mm parallel plate and an isothermal chamber. Inks
A and B were tested to evaluate the effect of Schiff-base 2.8.3. Cell morphology staining
formation on improving ink shape fidelity. Steady-shear MSCs were seeded onto scaffolds placed in a 24-well plate.
flow test was performed from 0.1 to 100 s at −1°С for each Actin-Tracker Red (Beyotime, China) and 4’6-diamidino-
−1
ink. An oscillation temperature sweep test was conducted 2-phenylindole (DAPI; Beyotime, China) were used for
from 37 to −5°С at a constant frequency of 1 rad/s and a immunofluorescence staining to visualize the cytoskeleton
strain of 0.5%. Lastly, a large amplitude oscillation sweep and nuclei, respectively. An upright fluorescence
(LAOS) test was conducted at a constant frequency of 1 microscope (DM6 B, Leica, Germany) was used for
rad/s across a strain range of 0.1–1000%. observation. The excitation/emission wavelengths were
496/516 nm for Actin-Tracker Red and 480/340 nm for
2.6. Thermal study DAPI, respectively.
Differential scanning calorimetry (DSC) test was
conducted on hydrogel ink A and B using a HITACHI 2.8.4. Western blot
DSC 200 instrument (HITACHI, Japan) under a nitrogen On days 7 and 21 of cell culture, protein was extracted from
environment. A cooling process from 30 to −10°С was MSCs. Cells were lysed in Radio Immunoprecipitation
applied at a rate of 1°С/min. Assay (RIPA) buffer (Beyotime, China), and total protein
concentration was determined using the Bradford assay
2.7. Mechanical study (Sangon Biotech, China). Protein of equal amounts was
Tensile tests were conducted on scaffold samples 1–4 using separated on a 12% Sodium Dodecyl Sulfate Polyacrylamide
a universal test machine (UTM 4103, Shenzhen SUNS (SDS-PAGE) Gel Electrophoresis gel and transferred onto
Technology Stock Co., Ltd., China). A loading rate of a 0.22 μm-pore size polyvinylidene difluoride membrane
1 mm/min was applied, and each group of scaffolds was (Sigma-Aldrich, USA) under a constant current of 252
tested in triplicate. mA. The membranes were then blocked with 5% skim milk

Volume 11 Issue 4 (2025) 227 doi: 10.36922/IJB025140116