Page 534 - IJB-9-5

International Journal of Bioprinting GelMA/PEG-TA IPN networks for 3D bioprinting

experiments. To avoid extrusion of the solution when were incubated at 37°C in humidified air (5 vol.%tw CO ).
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changing to the next layer, the piston of the syringe was The medium was refreshed every 2 days. The printing
retracted 0.5 mm after printing of each layer. Using these process with cells is graphically illustrated in Scheme 1.
conditions, scaffolds of 10 × 10 mm and a height of 5 mm The printed scaffolds were placed in culture medium, and
(20 layers) were printed. UV curing (365 nm) was set at a the samples were evaluated after 3, 7, and 21 days.
light intensity of 10 mW/cm with 5 s irradiation of each two
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layers during printing. Moreover, UV curing was applied 2.12. Live/Dead staining
for 1 min after completion of printing. Subsequently, the The live/dead viability/cytotoxicity kit was used to assess
GelMA/8PEGTA scaffolds were submerged in a 0.03 wt% cell viability. At scheduled time points, the cells were washed
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H O solution for 1 min to induce enzymatic crosslinking. gently with DPBS and 150 µL of a 2 µM calcein AM and
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The scaffolds were then submerged in PBS and incubated at 4 µM EthD-1 working solution was added directly to the
37°C overnight. 3D GelMA or GelMA/8PEGTA scaffolds cells. After incubation for 30 – 45 min at room temperature,
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with different geometries were printed for validation. the working solution was discarded completely. After
rinsing with warm DPBS, the sample was placed on a glass
2.10. Cell culture
slide to view the labeled cells with fluorescence microscopy
Osteosarcoma cells (MG-63 cells), which were selected as (3512001683, Carl Zeiss, Gottingen, Germany).
the model cell line, were cultured using a-MEM medium
supplemented with 10% (v/v) FBS and 1% penicillin and 2.13. Statistics analysis
streptomycin in an incubator at 37°C with 5% CO . The All data are expressed in mean ± standard deviation.
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culture medium was refreshed 3 times a week until the Biochemical assays were performed with triplicate
cells reached confluence. On confluence, the cells were biological sample, if not stated otherwise. Statistical
trypsinized and counted using a Neubauer cell counting analysis was performed using two-way analysis of variance
chamber. Cell suspensions with a concentration of (ANOVA) with Bonferroni’s multiple comparison test
1 × 10 cells/mL in culture medium were used for the (P < 0.05), unless otherwise indicated in the figure legends.
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preparation of bioinks. For all graphs, the following applies: *P < 0.05, **P < 0.01,
and ***P < 0.001.
2.11. Bioprinting of bioinks
Macromer stock solutions containing 12 wt% GelMA or 3. Results and discussion
12 wt% GelMA/4 wt% 8PEGTA were prepared using 3.1. Synthesis
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cell culture medium. The solutions were subsequently
disinfected using a pasteurization protocol by keeping The GelMA was prepared as previously described, and
them at 70°C for 30 min and then at 20°C for 30 min . approximately 90% of the lysine amine groups were
[42]
This procedure was repeated 3 times. To prepare bioinks converted into methacrylamide groups by reacting gelatin
[20]
containing cells, 2 mL of a macromer stock solution was with methacrylic anhydride . The 8PEGTA prepared
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gently mixed with 2 mL MG-63 suspension (1 × 10 cells/ by activation of the hydroxyl end groups with PNC and
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mL) at 37°C and transferred to a 5 mL syringe. The subsequent reaction with TA (Scheme 2) showed the
syringe was kept at 4°C for 10 min and mounted in the 3D appearance of aromatic protons at 6.79 and 6.99 ppm
bioprinter, the chamber temperature was set at 22°C, and (Figure 1). The degree of substitution of the 8PEGTA end
the system was incubated for 20 min. The syringe was set groups was determined by comparing the integral values of
to the starting position and printing was carried out using the aromatic protons with PEG protons, showing that five
the same procedure as given above for printing without out of eight hydroxyl end groups were substituted. Using
cells. Constructs of 10 × 10 mm and a height of 1 mm (4 this method, no full conversion of the hydroxyl end groups
layers) were printed in 6-well plates (n = 4). After printing, could be reached even by applying an excess of PNC.
all gels were post-crosslinked with UV (365 ± 5 nm,
10 mW/cm , 1 min). In addition, the GelMA/8PEGTA 3.2. Crosslinking
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gels were submerged in PBS containing 0.03 wt% H O Photo-crosslinking of GelMA and the enzymatic
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for 1 min and washed with PBS twice before fresh cell crosslinking of phenolic conjugates of synthetic and natural
culture medium was added. In the previous research, polymers are well-known methods to form hydrogel
we have shown that crosslinking of cell-laden tyramine- networks. Both crosslinking mechanisms are radical
conjugated natural polymers and 8-arm PEG using HRP at based. To show the independent crosslinking in macromer
low concentrations of H O shows no cytotoxic effects and mixtures, to form an IPN, by photoinitiation of GelMA and
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excellent biocompatibility [38,43] . The bioprinted constructs enzymatic crosslinking of the phenolic groups of 8PEGTA 5
Volume 9 Issue 5 (2023) 526 https://doi.org/10.18063/ijb.750