Page 255 - IJB-9-5

International Journal of Bioprinting

for the hydrogels soaked in 75% ethanol at the similar while S16 cells and HUVECs were used to initially explore
concentration and degree of methacryloylation. the delivery potential of the hydrogel for functional cells.
HUVECs, which are primary cells isolated from umbilical
There may be two main changes in the compressive cord veins, have a strong proliferative ability and good
properties after ethanol is treated. On the one hand, the performance, and could be induced to form vascular
SEM results (Figure 1D) showed that the ethanol-treated structures. S16 cells are the main glial cells in the peripheral
hydrogel was denser than the PBS-treated hydrogel, nervous system and could form myelin sheaths. S16 cells
and it was mainly nanoporous. Simultaneously, when could secrete neurotrophic factors, promote the survival
the concentration was higher, the material distribution of damaged neurons and the regeneration of axons, and
of the cross-section became tighter which caused the participate in the formation of nerve fibers in the peripheral
enhancement of compression performance. On the other nervous system. The results of the proliferation rates of
hand, the change of the secondary structure of ASF might the three types of cells, NIH/3T3, S16, and HUVECs, are
be an influencing factor. By analyzing the secondary presented in Figure 3A–C. All the three cells could proliferate
structure content, the ASF-MA formed hydrogel which normally on GelMA hydrogel, while BSF-GMA hydrogel
10%
caused an increase of the β-sheet content. It also helped was lack of the cell adhesion sequence RGD. Therefore, only
to enhance the compression performance, and the β-sheet low values were detected, and the proliferation could only
content would increase sharply after soaking in ethanol. be detected at day 7. However, cells could grow adherently
The increased β-sheet fraction implied that most of the on ASF-MA hydrogel, and the proliferation rate was close
molecules were in the crystalline state, which enhanced to that on GelMA hydrogel. According to the proliferation
10%
the hardness of hydrogel [32,33] . However, the compressive of the three cell types on the surface of ASF-MA PBS
10%
properties of the ethanol-soaked low-substituted ASF-MA hydrogels and ASF-MA EtOH hydrogels, although the
hydrogels became stronger. The possible reasons might be cell proliferation rate was relatively slow on the ethanol-
10%
that the small amount of MA had less effect on the ASF soaked hydrogels, the cells could proliferate normally.
secondary structure, and ethanol soaking had a higher
chance of β-sheet secondary structure transformation, The staining results are shown in Figure S1
leading to higher stiffness. (Supplementary File). The NIH/3T3 cells grew in the
clusters and spread all over on the GelMA hydrogel on day 5.
In Figure 2F, the 20% ASF-MA 10% EtOH hydrogel The cell proliferation rate on ASF-MA PBS hydrogel was
10%
achieved the highest tensile deformation of 65%, while faster than the rate on ASF-MA EtOH hydrogel, which
10%
ASF-MA EtOH hydrogel had stronger tensile strength. was consistent with the result of CCK-8. The cells on BSF-
5%
They could reach 686 kPa (54% deformation) and 830 GMA hydrogel were not adherent on the first 3 days, and
kPa (13% deformation) at 20% and 30% concentrations, some cells gradually adapted to the new environment and
respectively. However, when the tensile strength kept at started to spread around on day 5. However, when the plate
200 kPa, 20% ASF-MA 2.5% EtOH hydrogel showed greater was shaken vigorously, the cells could still be dislodged.
deformation, followed by 20% ASF-MA 10% EtOH hydrogel.
It might be caused by the increased β-sheet concentration As reported, S16 cells were sensitive to the mechanical
of the ethanol-treated hydrogels, which resulted in tighter properties of the substrate materials. If the basal materials
structure, less susceptibility to elastic deformation, were too soft or too stiff, it would not be conducive to the
stronger stiffness, and more brittleness. Thus, 20% ASF- spreading and growth of S16 cells . We observed the cell
[34]
MA 10% EtOH and 20% ASF-MA EtOH hydrogel showed states on four hydrogel materials. The results (Figure 3D)
5%
better tensile deformation. showed that the S16 cells grew in clusters on the GelMA
hydrogel, and the cells were still able to proliferate normally.
Taken together, we found that the different degrees For BSF-GMA hydrogel, fewer cells were attached, but
of methacryloylation and the different solution post- the number of proliferating cells increased significantly,
treatments would have impacts on the protein secondary which caused some single cells to spread into elongated
structure and would further affect the mechanical spindle shape on day 5. For ASF-MA hydrogel, S16
properties of the hydrogel. To meet the conditions of 3D cells could spread and grow individually. For ASF-MA
10%
10%
bioprinting with better mechanical properties of hydrogels, PBS hydrogel, most of the S16 cells spread into elongated
we decided to choose ASF-MA with the high degree of spindle shape, but a few cells grew in clusters on ASF-
10%
methacryloylation for follow-up research. MA 10% EtOH hydrogel.
3.3. Cytocompatibility of ASF-MA The growth condition of HUVECs on GelMA
The more commonly utilized NIH/3T3 cells were used to hydrogels was similar to that of NIH/3T3 cells (Figure S2
assess the cytocompatibility of the ASF-MA hydrogel, in Supplementary File). They were able to grow in
10%

Volume 9 Issue 5 (2023) 247 https://doi.org/10.18063/ijb.760