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International Journal of Bioprinting Enhanced osteogenesis in gelatin releasing bioink

3.6. Fabrication of MA-alginate/gelatin scaffold both the 5:5 and 7:3 MA-alginate/gelatin hydrogel scaffolds
After investigating the positive potential of remaining showed high viability; however, cells in the 5:5 MA-alginate/
gelatin in inducing bone regeneration activity of gelatin hydrogel scaffold showed higher proliferation than
encapsulated cells, the printability test for hydrogels was those in the 7:3 MA-alginate/gelatin hydrogel scaffold. This
performed to study their usage as bioinks. means that the remaining gelatin did have a positive effect
on the proliferation of encapsulated cells, reflecting the
MA-alginate/gelatin hydrogel samples with different
volume-to-volume ratios were prepared as per the earlier described results obtained with the hydrogel disks.
previous method to fabricate scaffolds using an extrusion These two types of scaffolds were also subjected to
3D printer. However, it was not possible to print with the Alizarin Red S staining after 3 weeks of differentiation, and
MA-alginate and 9:1 MA-alginate/gelatin hydrogels. Due the results are shown in Figure 9c and 9d. This stain is used
[17]
to the high content of alginate , these samples were to visualize calcium deposits, which is stained dark red.
too fluid and could not form appropriate struts before Calcium deposits were observed in both the 7:3 and 5:5
photo-crosslinking. Thus, the 7:3 and 5:5 MA-alginate/ MA-alginate/gelatin hydrogel scaffolds. However, it was
gelatin hydrogels were the only printable samples in our clear that 5:5 MA-alginate/gelatin hydrogel scaffold had
printing system. not larger area stained in darker red, as shown in Figure 9d,
compared to other scaffolds. Hence, 5:5 MA-alginate/
To determine the optimal ranges for the processing gelatin hydrogel scaffold had more cell mineralization than
parameters (pneumatic pressure and feed rate) for stable 7:3 MA-alginate/gelatin.
fabrication of high-fidelity scaffolds, a single line test was
performed. Figure 8a and 8b show the optimized ranges In summary, MA-alginate and gelatin were blended
for nozzle size, pneumatic pressure, and feed rate for in different volume-to-volume ratios to overcome the
producing stable struts. Different pressures were required limitations of single-component hydrogels and develop
to produce an accurate line for each nozzle size, and a biofunctional and printable hydrogel. Previous studies
pressures of 40–120 kPa were tested. The results shown were concentrated on modulating rheological properties
in Figure 8a illustrate the range of pneumatic pressures and mechanical properties of alginate–gelatin composite
required. As shown in Figure 8b, the strut width was by different solvents, various temperature settings or
dependent on using the optimal pressure for each nozzle different blending compositions for developing hydrogels
size, and this shows that it is possible to achieve the desired containing thermosensitive gelatin with higher printability
strut diameter. The 23G nozzle size was selected because it and extrusion uniformity [29-31] . Also, the application of
could be used to produce struts with a width of 404.65 ± cell-laden alginate–gelatin multi-component hydrogel as
34.23 μm. The moving speed, or feed rate, was evaluated bioink had been widely investigated. Recent studies were
to determine the range of printability. In brief, at a speed focused on keeping high cell viability by using different
of more than 300 mm·min , stable struts could not be concentrations of alginate and gelatin under required
-1
formed; non-continuous lines were produced. Through the pressures for optimizing printing conditions [33-37] . In these
printability test, the optimal range of fabrication conditions studies, more stable structures compared to those made
was determined. As a result, scaffolds were fabricated using of single components were printed, which had high cell
the cell-laden 5:5 and 7:3 MA-alginate/gelatin hydrogels viability during printing process but not cell differentiation.
with a pneumatic pressure of 80–100 kPa and a printing This indicated that gelatin was mostly suitable for printing
speed of 200 mm·min . stable structure and overcoming the limitation of alginate.
-1
The effects of gelatin on cells have been observed, but they
To confirm the stability of the encapsulated cells after are limited to cell proliferation [38-40] . Gelatin contains the
printing, live/dead assays were performed, and the results RGD sequence, which enhances the relationship between
are shown in Figure 8c and 8d. As shown in the image, cells and surrounding ECM. This may have a positive effect
many green-tagged cells could be observed immediately on the cell viability and proliferation.
after fabrication. This indicated that the encapsulated cells
were not critically damaged during the scaffold fabrication In this study, however, improving the printability of
process. alginate–gelatin multi-component hydrogels was not
our only focus. We paid more attention to the effects
3.7. In vitro cellular activities of the cell-laden of uncrosslinked gelatin, which was entrapped in the
hydrogel scaffold hydrogel. The key objectives of this study are: (i) to study
The cell-laden hydrogel scaffolds were cultured in the effects of gelatin released from the hydrogels on external
osteogenic differentiation media for 3 weeks, and then live/ cell, and (ii) to study the effects of gelatin remaining in the
dead assays were performed to evaluate the viability of the hydrogels on encapsulated cells. Therefore, we designed
encapsulated cells. As shown in Figure 9a and 9b, cells in the gelatin-loaded MA-alginate-based hydrogels. We

Volume 9 Issue 2 (2023) 152 https://doi.org/10.18063/ijb.v9i2.660