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International Journal of Bioprinting Enhanced osteogenesis in gelatin releasing bioink

the highest ALP activity levels at 1.08 ± 0.01 mM. This 0.03 μg/mL. This indicated that remaining gelatin in the
was significantly different from the amounts induced by MA-alginate/gelatin hydrogels had a positive effect on cell
the other gelatin-containing hydrogels (10:0 ratio: 0.76 ± proliferation, and the data presented in Figure 6a show that
0.02 mM, 9:1 ratio: 0.75 ± 0.02 mM, and 7:3 ratio: 0.89 ± the DNA quantity increased due to each type of hydrogel
0.05 mM). except the 10:0 MA-alginate/gelatin hydrogel.
Activation of osteogenesis in MC3T3-E1 cells was To further investigate the viability and spread of the
further investigated via gene expression analysis. qPCR was encapsulated cells, live/dead assays were used, and the
used to assess the relative expression of the genes encoding results are shown in Figure 6b. In this assay, live cells and
RUNX2, COL1, ALP, OPN, and OCN in cells 7 days after dead cells are tagged green and red, respectively. The cell
culture in osteogenic differentiation media, and the results viability test was performed after 3 weeks of culturing in
are shown in Figure 5c–g. Notably, for every marker gene osteogenic differentiation media. As shown in Figure 6b, it
evaluated—spanning early to mid-late stage osteogenesis— was difficult to detect dead cells in all the samples. However,
exposure to the hydrogel disk with a 5:5 MA-alginate/ the 5:5 MA-alginate/gelatin hydrogel was found to have
gelatin ratio resulted in the highest expression levels. In more live cells than the other hydrogels, and this finding
comparison to the gelatin-free MA-alginate hydrogel, the aligned with the DNA assay data shown in Figure 6a. The
5:5 MA-alginate/gelatin hydrogel induced the following live/dead assay was also used to assess cell spreading. It was
fold increases in expression: 2.32-fold for RUNX2, 2.16- only observed when the 7:3 and 5:5 MA-alginate/gelatin
fold for COL1, 2.43-fold for ALP, 1.28-fold for OPN, and hydrogel samples were used.
1.36-fold for OCN. Hence, the 5:5 MA-alginate/gelatin Overall, the DNA quantity was found to increase in
hydrogel and its associated released gelatin show potential cells included in every hydrogel type, except the 10:0
for having bone remodeling effects on cells.
MA-alginate/gelatin hydrogel. It was also found that cells
Based on the results from gelatin releasing profile included in the 7:3 and 5:5 MA-alginate/gelatin hydrogels
(Figure 4a), it was clear that higher amounts of gelatin were exhibited cell growth. This means that the encapsulated
released out from 5:5 MA-alginate/gelatin for entire period cells could spread in the hydrogel due to the empty spaces
compared to other groups because of higher amounts of left behind by the released gelatin, and that the remaining
gelatin entrapped during fabrication. Therefore, we could gelatin in hydrogel had a positive effect on cell viability and
conclude that for external cells, the higher amount of growth.
released gelatin, which was dissolved in the media, could
have positive affect on osteogenesis. 3.5. In vitro osteogenic behavior of encapsulated cell
After 3 weeks of culturing, the effect of the entrapped
3.4. Viability of cells encapsulated in the gelatin on the osteogenic differentiation behavior of the
MA-alginate/gelatin hydrogels encapsulated cells in each type of hydrogel was examined
On the other hand, the uncrosslinked gelatin, which by analyzing ALP activity and expression of osteogenic
was entrapped in hydrogels, remained even after 3 differentiation marker genes (Figure 7). Notably, the 5:5
weeks of releasing, as shown in Figure 4b. Additionally, MA-alginate/gelatin hydrogel group induced the highest
the enhancement of cellular activities of external cells ALP activity (1.37 ± 0.05 mM) compared to the other
caused by the released gelatin was confirmed, as shown groups (10:0 ratio = 0.13 ± 0.01 mM, 9:1 ratio = 0.33 ±
in Figure 5. Therefore, the activities of encapsulated cells 0.01 mM, and 7:3 ratio = 0.43 ± 0.03 mM). This indicated
influenced by the remained gelatin in each hydrogel were that the remaining gelatin in the 5:5 MA-alginate/gelatin
also investigated. hydrogel had a stronger effect on cell differentiation via the
The effect of gelatin entrapped in the hydrogel on increase in ALP activity than that remaining in the other
cell proliferation of the encapsulated cells was analyzed three types of hydrogel.
by measuring the amount of DNA, and the results are The effect of the remaining gelatin on osteogenesis was
shown in Figure 6a. Throughout the culturing period, also examined by gene expression analysis, and the results
the cells within the gelatin-containing hydrogels showed are shown in Figure 7b–f. The selected marker genes have
higher quantities of DNA than those within the gelatin- been used in previous studies. Regarding the expression of
free hydrogels. After 3 weeks, the hydrogel disks with a 5:5 the RUNX2, COL1, and ALP genes, the 5:5 MA-alginate/
MA-alginate/gelatin ratio induced the highest quantities gelatin hydrogel induced significantly higher levels of
of DNA, with a mean of 1.31 ± 0.06 μg/mL. The other each compared to the other hydrogels, as shown in Figure
hydrogels produced the following DNA quantities: MA- 7b–d. It induced the following fold increases compared to
alginate = 0.42 ± 0.01 μg/mL, 9:1 MA-alginate/gelatin = the MA-alginate hydrogel: 8.31-fold for RUNX2, 16.01-
0.91 ± 0.03 μg/mL, and 7:3 MA-alginate/gelatin = 0.99 ± fold for COL1, and 12.84-fold for ALP. Moreover, the

Volume 9 Issue 2 (2023) 149 https://doi.org/10.18063/ijb.v9i2.660