Page 556 - IJB-10-3

International Journal of Bioprinting Engineered 3D-printed PVA vascular grafts

cells, following a 24-h exposure to the biomaterial extract displaying unchanged cell shape or density relative to the
in cell culture medium and extract dilutions, was measured negative control (Figure 12C–F).
using the MTT assay. The metabolic activity of the cells A cytotoxic effect is induced by the extract of the
is a direct measure of their viability and an indicator of material in the cell culture of the initial vascular graft in the
vascular graft biocompatibility. Viability above 70% is presence of bEnd.3 endothelial cells. A cell culture medium
considered a threshold for biocompatibility, according to wash was subsequently applied to the graft to evaluate the
ISO 10993-5:2009. direct contact effect on the endothelial cells. Following this

The viability of the endothelial cells exposed for 24 h washing step, the cytotoxic residues were removed from
to undiluted vascular graft extract decreased by 40.21 ± the graft, resulting in a biocompatible effect.
4.25% compared to the control cells, resulting in a slightly
cytotoxic behavior (p < 0.05) (Figure 11A). A similar effect 3.9.3. Biocompatibility assessment of the modified
was obtained for the 1:2 diluted extract in cell culture vascular graft using the extract method
media, whereby the cells had a viability of 58.97 ± 0.58% Additionally, the material was functionalized using lysine
compared to the control cells (p < 0.001) following a 24-h to increase the biocompatibility of the graft for cells in the
incubation period (Figure 11A). LDH release (Figure 11B) vascular environment. The resulting modified vascular
from cells exposed to undiluted extract and 1:2 diluted graft biocompatibility was assessed for endothelial cells,
extract reported a reduced amount compared to the control fibroblasts, and monocytes to determine the metabolic
cells (p < 0.05), indicating that necrosis is not the induced behavior (MTT and ATP assays) and integrity (LDH assay)
cell death pathway for bEnd.3 cells. Instead, this reduction of the cells.
suggests a decrease in cell number, evidenced by the slight The modified graft 3D-3H-lysine displayed improved
reduction in cell density in the optical microscopy images biocompatibility compared to the initial vascular graft for
(Figure 11D and E). bEnd.3 endothelial cells, with a viability of 77.8 ± 2.56%
This slight cytotoxic effect can be attributed to the compared to the negative control (p < 0.01) (Figure 13A).
release of weakly attached GA molecules that were not ATP production in the graft extract-treated endothelial cell
incorporated into PVA via crosslinking. In contrast, 1:5 culture was above 80% compared to the negative control
and 1:10 diluted extracts reported biocompatibility (cell (p < 0.01) (Figure 13B). LDH release is lower than the
viability was approximately 100%) with similar LDH negative control, i.e., at 0.157 ± 0.051 compared to non-
release compared to the negative control (Figure 11A and treated cells (p < 0.05). This could be due to a reduction
B). Cellular morphology and density were also similar to in cell number and interference between the extract
the non-treated controls (Figure 11C, F, and G). compounds and LDH (Figure 13C). However, optical
microscopy observations did not reveal any obvious
3.9.2. Biocompatibility assessment of initial vascular morphological and cell density changes in the treated
grafts using the direct contact method bEnd.3 cells compared to negative control (Figure 13D and
To simulate the endothelial cells’ direct contact with the G).
initial vascular graft, an endothelial cell monolayer was The effect of the modified vascular graft extract
seeded and grown for 24 h prior to the addition of the on fibroblast cells also reported biocompatibility
vascular graft sample. For this, samples of the graft were compared to the negative control, with cell viability of
cut to have similar dimensions. Additionally, the samples approximately 100% (Figure 13A). However, a reduction
were soaked and incubated overnight in a complete in fibroblasts’ ability to produce ATP was noted, i.e., 29.71
culture medium to eliminate any cytotoxic residues from ± 0.6% compared to non-treated cells (p < 0.01) (Figure
the biomaterial extract. Thereafter, the graft samples were 13B). Generally, the MTT assay evaluates the overall
directly added on top of the cell monolayer and incubated mitochondrial activity of the cells, while the ATP assay
for another 24 h in standard conditions.
is a quantitative measure of the ATP molecules, which
Viability assay measurements displayed a slight store the cell’s energy. ATP measurements validated the
reduction of the cells’ metabolic activity with 13.47 ± biocompatibility of the vascular graft extract on L929
10.9% relative to the negative control (Figure 12A), cells (ISO 10993-5). A small amount of extracellular LDH
indicating biocompatibility according to ISO 10993- was quantified, albeit not significant compared to the
5:2009. Additionally, LDH release reported no significant negative control (Figure 13C). Cellular morphology was
differences compared to the negative control (Figure 12B). not affected by the treatment with undiluted graft extract.
These results were confirmed by morphological evaluations However, a slight reduction in cell density compared to
of the cell monolayer following incubation with the graft, negative control was noted (Figure 13E and H).

Volume 10 Issue 3 (2024) 548 doi: 10.36922/ijb.2193