Page 259 - IJB-9-5

International Journal of Bioprinting

well. For the GelMA and BSF-GMA hydrogels, S16 cells For all three GelMA, BSF-GMA, and ethanol-soaked ASF-
were in a non-spreading circular state, and the F-actin was MA 10% hydrogels that were under the same cell density, we
more distributed in the edge part and less distributed in observed that some cells on each hydrogel grew in clusters.
the inner part of the cells. The fluorescence intensity would Thus, we came to the conclusion that the increased
become higher when the cells were denser. It showed intercellular contact area would impact the expression of
no significant difference in the average fluorescence PMP22, which had less effect on MPZ protein [5,45-47] . Based
intensity of individual cells. For vinculin, the single cell on these results, it could be tentatively concluded that ASF-
fluorescence intensity was observed under a microscope MA 10% PBS hydrogel could promote adhesion, migration,
and incorporated for the fluorescence semi-quantitative and myelination of S16 cells.
analysis (Figure 5B). We noticed that a higher expression
of vinculin was observed on GelMA and ASF-MA 10% 3.7. In vitro and in vivo degradation test
hydrogels, and fluorescence was observed in S16 cells To evaluate the potential of hydrogels as implant
on ASF-MA 10% PBS hydrogels in some areas. In contrast, materials in vivo, we performed in vitro degradation and
S16 cells on BSF-GMA hydrogels showed less vinculin subcutaneous implant degradation experiments on three
expression and diffused fluorescence. All of the results different hydrogels. The results of in vitro degradation are
suggested that the ASF-MA 10% PBS hydrogel was beneficial shown in Figure 6A and B. The obviously slow degradation
to the adhesion and the spreading/elongation of S16 cells, rate in the upper part of the figure was the hydrogel in
while the ASF-MA 10% EtOH hydrogel did not show any PBS, and the faster degradation rate in the lower part was
other obvious advantages or disadvantages. the hydrogel in PBS containing protease XIV. We could
clearly see that the degradation rate of the ASF-MA PBS
3.6. Expression of related genes in S16 cells hydrogel was faster, and the degradation rate of the ASF-
S16 cells are the main glial cells in the peripheral nervous MA EtOH hydrogel was obviously slowed down, regardless
system and can form myelin sheaths. We selected several of whether the hydrogel was in the PBS solution or the
proteins related to the adhesion, motility, and myelination of solution containing protease. This might be caused by
S16 cells to further explore different effects of hydrogels on increased β-sheet content and increased crystallinity .
[32]
the growth of S16 cells. In Figure 5C, the relative expression
of four genes related to cell adhesion (Integrin β1, Cdc42, As shown in Figure 6C and D, the three hydrogel
Rac1, and NCAD) under the ASF-MA 10% PBS hydrogel materials gradually degraded under the mice’s skin. The
condition was significantly higher than that under the GelMA hydrogel was slightly swollen at week 1 and week 4
BSF-GMA hydrogel condition. The expression of Integrin with 26% degradation at week 12 . The BSF-GMA hydrogel
β1 and Rac1 under ASF-MA PBS hydrogel condition followed the similar pattern that was swollen at week 4 and
10%
was lower than that under GelMA hydrogel condition. then gradually degraded with 25% degradation at week 12.
Nonetheless, the expression of Cdc42 and NCAD under Compared with the GelMA hydrogel and the BSF-GMA
ASF-MA 10% PBS hydrogel condition was not significantly hydrogel, ASF-MA hydrogel degraded faster with 27% at
different from that under GelMA hydrogel condition. The week 4. The degradation reached 32% at week 12, which
expression of these four genes under ethanol-soaked ASF- was slightly higher than that of the first two hydrogels.
MA 10% hydrogel condition was slightly lower than that We speculated that it might be related to the higher
under PBS-soaked hydrogel condition, but they showed temperature of the regenerated ASF, which made it easier
no significant differences. Once again, this indicates that for molecular chain breakage to occur, so the ASF-MA was
ASF-MA 10% PBS hydrogel could promote S16 cell adhesion more susceptible to degradation.
and migration, which was comparable to that on GelMA The results of hematoxylin and eosin (H&E)
hydrogel and significantly better than that on BSF-GMA section staining shown in Figure 6E suggested that the
hydrogel. The expression of the MPZ gene, which encodes inflammatory response following the subcutaneous
a protein related to myelination, was higher on ASF-MA GelMA hydrogel implantation was weaker. At week 1, the
10%
PBS hydrogel compared to the other three hydrogels and inflammatory response was more substantial, and the outer
showed differences. However, the expression of PMP22 layer of hydrogel was infiltrated with more immune cells,
gene was decreasing on GelMA, BSF-GMA, and ASF- although it gradually weakened after 2 weeks. ASF-MA 10%
MA 10% PBS hydrogels, and it gave a significant difference had a weaker immune response at week 1 of implantation,
as a result. The S16 cell line used in our study provided which gradually became stronger afterward. By week 8,
the characteristics of high myelinated protein expression, the inflammatory response gradually weakened, and some
and the expression of PMP22 decreased sequentially fibroblasts appeared in the outermost layer. In contrast,
on the three hydrogels which might be due to the cell the inflammatory response of BSF-GMA hydrogel was
density that could affect the expression of PMP22 [41-44] . more substantial and tended to diminish at week 8. While

Volume 9 Issue 5 (2023) 251 https://doi.org/10.18063/ijb.760