Page 476 - IJB-10-2

International Journal of Bioprinting Bioprinting with ASCs and bioactive glass

after 14 days in culture to handle and perform biological mL) could be toxic to ASCs, whereas low concentrations
assays. In general, hydrogel degradation depends on the alter ASC protein secretions that may regulate wound
soaking media composition and enzymes present in it. In healing. Several studies have investigated the toxicity of
our study, DI water was utilized to prepare all hydrogels and silicate-based bioactive glasses toward stem cells in a 2D
to investigate the scaffold swelling characteristics, which environment, reporting that an optimum concentration
are different from other studies where PBS or DMEM was of ionic dissolution products could exist upon which the
utilized. Nevertheless, samples used for mechanical tests treatment that could be toxic to cells was administered. 44-46
were stored in DMEM to mimic the in vitro environment. To establish a baseline, we first investigated the B3 glass
In addition, we also investigated the structural integrity of toxicity to ASCs in 3D environment by adding glass in
2.5G scaffolds in the CCM at culture conditions without different weight percentages to AG hydrogel. First, ASCs
crosslinking with CaCl solution. This test was performed were encapsulated in AG, 1.25G, 2.5G, 5G, and 10G
2
to examine if the divalent cations (Ca and Mg ) that are hydrogels, and 3D-bioprinted as spheroids. To investigate
2+
2+
released from B3 glass into the hydrogel, which helps initiate the cell viability, spheroids were cultured in both static
the hydrogel crosslinking, would be sufficient to sustain the (6-well plates under standard culture conditions) and
scaffold structural integrity in culture conditions. Figure 9 dynamic (6-well plates kept on a rocker) conditions. Figure
shows a collapsed 2.5G scaffold that was not crosslinked 10 shows the live/dead assay images of the spheroids on
after a 2-h incubation in CCM at 37°C, whereas a scaffold day 0 (2 to 4 h after extrusion and crosslinking), day 1,
crosslinked with 0.1 M CaCl retained its structural fidelity and day 4 after culture. Spheroids made with 5G and 10G
2
after 7 days in culture. This result demonstrates that despite spheroids were irregular in shape because of the high
the improvement achieved with B3 glass addition in terms viscosity, and difficulties were encountered to uniformly
of printability of AG hydrogel, rheological characteristics, mix ASCs in these hydrogels. Patches of the hydrogel with
and initial mechanical properties, addition of glass alone empty pockets (without cells) and irregular shapes could be
would not be sufficient to fabricate a 3D scaffold without observed in the live/dead images of 5G and 10G hydrogels
the chemical crosslinking using CaCl solution. (Figure 10d, e, i, and n). Also, significant number of dead
2
cells (red spots) can be observed in the live/dead images of
3.3. Effect of B3 glass on ASCs viability 5G and 10G spheroids at all time points which was not the
One important objective of this study was to evaluate the case with other spheroids, whereas more viable ASCs than
amount of B3 glass that is acceptable to add to the AG dead cells were noticed in AG, 1.25G, and 2.5G spheroids.
hydrogel to provide viable human ASCs. As B3 glass dissolves
much faster than traditional silicate-based bioactive glasses Figure 11 shows the quantification of the live/dead
and faster than 45S5 glass, it was important to establish that assay results. The results clearly indicate that increasing
the resultant concentration of ionic dissolution products the percentage of B3 glass could be toxic to ASCs in the
has no toxic effects on ASCs in vitro. The toxicity could hydrogel. The viability of ASCs in AG spheroids without
be resulted due to pH increase as alkali ions are released B3 glass was significantly higher than ASC viability in
to CCM with B3 glass dissolution. Recently, B3 glass was 10G, 5G, and 2.5G spheroids immediately after extrusion
added to ASCs in two different approaches: directly being and crosslinking with CaCl . ASC viability in spheroids
2
added as particles during cell culture, and indirectly (glass cultured under dynamic conditions was also higher in
dissolution products) exposing cells to two-dimensional comparison to viability in spheroids cultured under static
(2D) cell culture environment at a B3 glass concentrations conditions after 24 h. This could be due to efficient nutrient
that are less than 10 mg/mL. 30,35 These studies reported transfer to ASCs under dynamic conditions than static
that a high concentration of B3 glass exposure (>10 mg/ conditions. In addition, for spheroids containing B3 glass,
Ca , B , and other ions released after glass dissolution in
2+
3+
the spheroid could diffuse more freely to the media under
dynamic conditions than static culture conditions. This
could cause the pH of the spheroid to go basic in static
conditions and negatively affect ASCs. Addition of B3
glass increased the pH of hydrogels from neutral for AG to
pH 8 for 1.25G, pH 8.5 for 2.5G, and up to pH 9 for 10G
hydrogel. This could be the reason for high toxicity of B3
glass to ASCs, especially in 2.5G, 5G, and 10G spheroids
on day 0, i.e., immediately after mixing ASCs. Though the
inside of spheroid maintained relatively high pH, the pH
Figure 9. 2.5G scaffold in CCM at 37°C after 2 h (left) and after 7 days of the surrounding CCM remained neutral during culture
(right).

Volume 10 Issue 2 (2024) 468 doi. 10.36922/ijb.2057