Page 31 - IJB-9-1

International Journal of Bioprinting Biocompatible materials and Multi Jet Fusion

those on the uncoated surface in comparison to other appeared and survived on seeding. Nonetheless, the electron
coating or plasma-treatment, as determined at day micrographs provided additional evidence showing the
4 timepoint. On the other hand, MC3T3e1 osteoblasts presence and adherence of cells on the printed PA-12. In
grew better on PDL-coated 3D-printed PA-12 (P < 0.05) contrast, a completely different behavior was observed for
and CLG-coated polystyrene plate (P < 0.05). These osteoblasts which were bigger in size (>50 µm) compared
observations indicated that PDL was the preferred to fibroblasts. Osteoblasts exhibited a flattened shape and
choice as an extracellular matrix for culturing cells of appeared to spread over multiple particles and had many
both types on 3D-printed PA-12, even though it did not prominent filopodia protrusions stretching over multiple
substantially narrow the gap with polystyrene plate. The particles (Figure 4B i–iii). As a result, the main cell body
fact that fibroblasts survived better in the 3D-printed appeared loosely attached and suspended between particles,
PA-12 cell culture chambers than osteoblasts during making it difficult for the osteoblasts to completely attach
short-term culture was apparent. Cells demonstrated and proliferate. Control PA-12 cell culture chambers
equal proliferation on uncoated hydrophobic PA-12 (medium only) are also shown for reference (Figure
surfaces, even in the absence of surface coating or O S11A i–iii and S11B i–iii). Fibroblast and osteoblasts cells
2
plasma-treatment, indicating that the surface wettability (and control samples without cells) grown on polystyrene cell
did not significantly affect the cell adherence. When culture chambers are also shown for comparison (Figure S11C
substrates were plasma-treated, a significant decrease in i–iii and S11D i–iii). Taken together, these findings supported
hydrophobicity was observed (P < 0.05). Even though the fact that 3D-printed PA-12 can support cell growth, but not
substrates demonstrated higher wettability, this did not to the extent observed on polystyrene plates, probably due to
significantly improve the cell adhesion on the substrates. the microroughness resulting from partially fused particles on
Therefore, hydrophobicity was not considered a key factor the surface among other factors.
in determining the biocompatibility of 3D-printed PA-12.
3.3.3. Cellular toxicity
Representative SEM images of L929 and MC3T3e1 cells
cultured on 3D-printed PA-12 and polystyrene cell culture The short-term exposure of L929 fibroblasts and MC3T3e1
chambers are shown in Figure 4A–D. In all the experiments, osteoblasts to uncoated, PDL- and CLG-coated 3D-printed
L929 fibroblasts and MC3T3e1 osteoblasts cultured on PA-12 cell culture chambers, and polystyrene plate resulted in
coated and uncoated 3D-printed PA-12 cell culture chambers minimal LDH secretion due to cellular membrane integrity
presented a flat morphology and appeared intimately adherent on day 2 and day 4, respectively. Consequently, cells cultured
to the surface, with no specific orientation. L929 fibroblasts on both coated (PDL and CLG) and uncoated 3D-printed
and MC3T3e1 osteoblasts cultured on coated and uncoated PA-12 demonstrated no signs of cytotoxicity. No significant
3D-printed PA-12 cell culture chambers appeared intimately difference in cytotoxicity was observed between coated and
adherent to the surface and showed an increase in the number uncoated substrates, as shown in Figure 5A and B.
of cells attached to the surface, noticeable after 4 days of 3.3.4. Redox characterization
incubation. Fibroblasts exhibited cellular dimensions (~15 µm
in diameter) (Figure 4A i–iii) smaller than the unfused Visualization of oxidative stress assessed by mBCI staining
PA-12 particles, making it conducive for cells to adhere to a of L929 fibroblasts and MC3T3e1 osteoblasts cultured on
single particle. Interestingly, unlike in the fluorescent images, 3D-printed PA-12 cell culture chambers and polystyrene
the morphology of L929 grown on PA-12 and polystyrene plate showed preservation of intracellular reduced GSH on
appeared different in the SEM images. Such differences could both day 2 and day 4, regardless of the type of surface coating
be devoted to intrinsic artifacts such as specimen shrinkage (uncoated and coated) (Figure 5C and Figure S12). For
[55]
more likely as an effect of glyceraldehyde fixation or ethanol fibroblasts, GSH-mBCl fluorescence signal was comparable
[56]
dehydration before critical point drying. Some earlier between cells grown on 3D-printed PA-12 cell culture
[57]
studies had also suggested great variation of these artifacts chambers and polystyrene plates at both time points,
from one type of sample to another . It was also indicated suggesting that cells were not significantly stressed on
[58]
that the use of high-resolution SEM had known to accentuate culture in 3D-printed PA-12 cell culture chambers. However,
artifacts which might otherwise appear minimal at a lower osteoblasts cultured on 3D-printed PA-12 showed a reduction
resolution . In addition, the influence of the MJF-printed in the overall intensity when compared to that observed on
[59]
PA-12’s surface properties (such as roughness) on preserving polystyrene plate, possibly as a result of the lower cell count.
cell appearance from the effect of SEM-processing methods
had to be highlighted. Moreover, the literature had minimal 3.3.5. Cellular proliferation
evidence on the effect of critical point drying on 3D-printed The proliferative state of cells characterized by
PA-12, which had a major role in deciding on how the cells immunostaining of Ki67 and p53 suggested that

Volume 9 Issue 1 (2023) 23 https://doi.org/10.18063/ijb.v9i1.623