Page 190 - IJB-9-2

International Journal of Bioprinting A regulated GelMA-MSCs scaffold by three-dimensional bioprinting

The changes of the surface defect of distal femoral condyle 3. Results
were quantitatively evaluated by coronal and sagittal
reconstruction analysis, and ImageJ software was used to 3.1. Characterization of GelMA scaffolds
quantitate the surface defect repair. GelMA scaffolds can provide a suitable 3D environment for
cell growth, proliferation, migration, and differentiation,
2.5.4. Micro-CT analysis which are crucial for further nutrient delivery and defect

Excess soft tissue and bone were removed from proximal repair. In this study, a photo-cross-linked GelMA scaffold
and distal regions of the femoral condyle. Samples were with high biocompatibility, acceptable mechanical
fixed in 4% paraformaldehyde fixative (sample size = strength, and high swelling was selected. A comparative
18). Next, samples were scanned using microcomputed study was conducted between GelMA-60 5% and GelMA-
tomography (micro-CT, Skyscan 117, Basserdorf, 90 10%. It was found that the Young’s modulus of GelMA-
Germany) with a slice resolution of 18 μm. Micro-CT 90 10% was significantly higher than that of GelMA-60 5%
slices were imaged at an X-ray energy level of 70 kVp and (Figure S1A). By measuring the pore size of the two
a current of 114 μA with an integration time of 150 ms. scaffolds, it was found that the 5% pore size of GelMA-
After scanning, data were exported in NRecon software 60 was 176 ± 26.34 μm, which was larger than the 10%
for 3D reconstruction. CTAn and Mimics 10.01 software pore size of GelMA-90 (94 ± 14.23 μm) (Figure S1B).
extracted volumetric and densitometric numbers from the Larger pore sizes can create larger spaces. It is beneficial
gray value distribution of the 3D-reconstructed images. to cell growth, proliferation, and migration. Considering
that the scaffold may fall off during the movement of
2.5.5. Immunohistochemistry staining and analysis rabbits after implantation of the defect, the appropriate
Isolated femoral condyle specimen of rabbits fixed swelling rate of the scaffold can make itself better fit in
with 4% paraformaldehyde for 3 days. Subsequently, the defect. By measuring GelMA scaffolds in two different
they were decalcified in 10% EDTA buffer for about concentrations, it can be found that the swelling rate of
2 weeks, dehydrated in graded alcohol, and embedded GelMA-60 5% scaffolds is superior to the GelMA-90 10%
in paraffin, and the slice thickness was controlled at scaffolds (Figure S1C), which is probably due to the larger
3 μm. The sections were stained with hematoxylin-eosin pores of GelMA-60 5% scaffolds, and the water absorption
(HE), Masson’s trichrome, and Safranin O fast green rate is also naturally higher, resulting in better swelling
(S-O FS) stains to observe cell morphology. At the same compared to the GelMA-90 10% scaffolds.
time, the expression of Col II and BMP-2 was detected Based on our observation (Figure S1D and S1E),
by immunohistochemical (IHC) method using mouse GelMA-60 5% scaffolds had a large aperture and were
anti-rabbit Col II and BMP-2 antibodies. The repaired loose as a whole. Too high GelMA concentration results in
tissue was evaluated using a modified Wakitani grading the formation of excessively dense cross-linked structure
system consisting of five categories: cell morphology, with increased hardness and decreased pore size, which
matrix staining, surface regularity, cartilage thickness, adversely affects cell viability within the scaffold structure.
and integration of the donor with the host. As previously Combined with the characterization and detection of
reported, cell morphology was graded using a scale from GelMA scaffolds in this study, it is preliminarily believed
0 to 4; matrix staining, surface regularity, and cartilage that cellular compatibility and cellular activity play a more
thickness, on a scale from 0 to 3; and integration of the important role in the repair of full-layer cartilage. In this
donor with the host, on a scale from 0 to 2 – with a perfect aspect, GelMA-60 5% is more suitable for repairing full-
total score being 15 points. thickness cartilage defects.
2.6. Statistical analysis 3.2. Multi-lineage differentiation and identification

Statistical analyses were performed using the Statistical of MSCs
Package for the Social Sciences (SPSS 19.0, Chicago, IL). A large number of adherent-growing rabbit bone marrow
All data are expressed as mean ± SEM. The Levene’s test was MSCs were obtained through primary culture and
first performed to check the normality of the distribution subculture. 3 h after inoculation, the cells began to adhere
and determine, whether the equal variance assumption of to the bottom of the culture dish. The cells were cultured
the data was violated. Multiple group comparisons were for 5 days, and the medium was changed every 48 h. The
performed using one-way analysis of variance (ANOVA) majority of the cells were in the shape of a helical growth
to evaluate the significance of the experimental data. fusiform, and merely, a few cells were in a triangular shape.
A confidence level of 95% (P < 0.05) was considered After three passages, a morphologically homogeneous
significant. population of fibroblast-like cells was observed. Even after

Volume 9 Issue 2 (2023) 182 https://doi.org/10.18063/ijb.v9i2.662