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International Journal of Bioprinting Enhanced osteogenesis in gelatin releasing bioink

Figure 1. Osteogenesis enhanced alginate–gelatin-based bioink by uncrosslinked gelatin releasing and retaining.

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2.3. Cell culture cells at a concentration of 5 × 10 cells/mL were added to the
The mouse calvaria-derived preosteoblast cell line mixed solutions homogenously, and the resultant mixtures
MC3T3-E1 (American Type Culture Collection, USA) was were stored at 4°C for 30 min. The mixed solutions were
cultured in alpha-minimum essential medium (α-MEM; subjected to ultraviolet-light irradiation (365 nm) for 1 min
Gibco, USA) with 10% fetal bovine serum (Gibco, USA) at room temperature for photo-crosslinking and prepared
and 1% penicillin-streptomycin (Gibco, USA) at 37°C as disks (1 mm height, 8 mm diameter). After fabrication,
with 5% CO . Culture media was refreshed every 2–3 days. the groups of hydrogel disks (n = 4) were transferred to 24-
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After rinsing the cell culture once with phosphate-buffered well plates for culturing.
saline (PBS), cells were harvested with 0.25% trypsin-
ethylenediaminetetraacetic acid (EDTA) treatment for The cell-free hydrogels were prepared under the same
4 min, followed by centrifugation for 3 min at 1,500 rpm. conditions, but the addition of cells was excluded. These
cell-free hydrogels were cultured on the top of transwells
2.4. Preparation of MA-alginate/gelatin mixture for proving gelatin releasing. For transwell system, the
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solution and hydrogel harvested cells at a concentration of 2 × 10 cells/mL
MA-alginate was dissolved in PBS containing the photo-initiator were seeded on 24-well plates and cultured under gelatin
0.05% lithium phenyl-2,4,6-trimethylbenzoylphosphinate releasing condition.
(LAP) to achieve a concentration of 2% (w/v). Gelatin was For the osteogenic differentiation study, the hydrogel
dissolved in the same buffer solutions as MA-alginate at 37°C disks that contained cells were cultured for 3 weeks in
at a concentration of 6% (w/v). The resulting solutions were osteogenic differentiation media. On the other hand, the
mixed together at different volume-to-volume ratios (10:0, cell-free hydrogels which cultured cells on the well plates
1:9, 5:5, and 7:3) at 37°C overnight.
were cultured for 1 week in osteogenic differentiation
To encapsulate cells in the hydrogel disks with different media. This was made from culture media by adding
MA-alginate/gelatin volume-to-volume ratios, harvested 50 μg/mL L-ascorbic acid 2-phosphate sesquimagnesium

Volume 9 Issue 2 (2023) 144 https://doi.org/10.18063/ijb.v9i2.660