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Biocompatible materials and Multi Jet Fusion

high cell assembly on PA-12 particles (Figure 4A). On the on 3D-printed PA-12 cell culture chambers that are actively
other hand, osteoblasts are mostly irregularly flattened proliferating cells and remain unaffected. The expression
with elongated filopodia extensions roughly equally from of the nuclear Ki-67 antigen is strictly associated with
each side of the cell that appears extensively spread. The cell proliferation and not present in resting or quiescent
uneven PA-12 surface and the large size of osteoblasts cause cells, in which many proliferation-related proteins remain
the cells to stretch/extend over many unfused particles. degraded . As a useful predictor for recognizing rapidly
[68]
This facilitates the attachment of the filopodia extensions proliferating cells, Ki-67 is constitutively expressed by
causing a “pulling effect” on the cell edges, and therefore, fibroblasts (Figures S13–S15). However, studies indicate
the main cell body appears unattached and suspended from that it may also be present in low levels in some cells (like
the PA-12 substrate (Table 1). Moreover, the rougher PA-12 osteoblasts as observed in Figures S16–S18) .
[69]
surface makes it difficult for osteoblast to reach confluence. During in vitro cell culture, long-term successive
Hence, a stable surface attachment evidenced in fibroblasts cell passaging is inevitable. Even though the osteoblast
is not seen in osteoblasts, making it difficult for them to
proliferate in the same way. In contrast, cells grown on adherence and morphology are sustained in prolonged
polystyrene substrates are well adherent, proliferative and cultures (Figure 7A), the viability and proliferation
particularly attain confluence (Figure 4D i–iii). However, ability are greatly compromised in MC3T3e1 osteoblasts
cell morphology with spread and expanded cytoskeleton after continuous passaging (Figure 7C). While culturing
remained similar to that observed on PA-12. The osteoblast MC3T3e1 osteoblasts, the passage number of the cells
morphology, proliferation, and metabolic activity seem to matter as they can influence cell function. Cells at passage
be more sensitive to the topography of the substrate. The 25 are used for this study. Researchers advise against the
results suggest that the surface roughness of MJF-printed use of osteoblast cells above 30 passages which involve
[70]
12 has a differential effect on the proliferation of different few risks that occur beyond safe passage . Moreover,
cell types. It negatively affects MC3T3e1 more than L929, cells are revived from a -80°C freezer during which the
possibly due to the larger cell size of the former. Since cell physiological activities are maintained at a relatively
adhesion is a necessary step in promoting cell growth, lower level. It might take a couple of passages for the cells
[71]
the imperfect contacts between MC3T3e1 and the surface to recover in terms of functionality . It is no surprise
could have weakened the transduction of growth signaling that the osteoblasts are less functional given their retarded
pathway from the extracellular matrix to the cells. This can proliferative behavior under normal circumstances when
be addressed by polishing the surface of printed PA-12 cultured on 3D-printed PA-12 cell culture chambers as
with sandpaper to smoothen out all the irregular globular aforementioned. The same phenomenon is also observed
bumps. Alternatively, the surface could be coated with a in positive controls (Figure 7C). When using osteoblast
layer of biocompatible polymer, such as polystyrene, to form cells, such complications during culture may arise when
a smooth even surface for attachment. Smoothening the considering interspecies differences, primary versus
surface is expected to enhance not just MC3T3e1 but also established cell cultures, and the possibility of phenotypic
L929, attachment, and proliferation. To further characterize heterogeneity of osteoblastic cells when obtained from
[72]
the effect of roughness on cell growth, a combination of different anatomical sites .
other evaluation assays is included to further test the in Unlike MC3T3e1, there is no evidence in our results
vitro cytotoxicity. suggesting that PA-12 impaired the L929 fibroblast’s ability

The LDH assay is another commonly used marker for to proliferate from subculture to subculture. However,
cell death. Negligible LDH release is observed in both certain morphological abnormalities were also observed
cell lines (Figure 5A and 5B), indicating no cell death nor mostly due to the surface topography of different substrates
cell membrane damage as the cells continue to proliferate indicating the fact that even though cells adhere to the
after day 4 (as suggested by long-term culture studies, substrates, they may not have completely adapted to the
Figure 7A–C and Figures S20–S25). Moreover, the influence topography of its surface (Table 1). Of note, cells transferred
of oxidative stress in causing cytotoxicity is correlated to the from PA-12 to plate retain the ability to grow as robustly
upregulation or downregulation of various gene regulatory as those subcultured from plate to plate. PA-12, especially
proteins. Since the intracellular GSH levels are significantly the coated ones, did not impair their ability to grow on
higher (as represented by high mBCI fluorescence signals conventional culture plates in subsequent subculture
in Figure 5C and Figure S12), corresponding levels of (Figure 7B). This is important firstly because we do not
proliferative marker (Ki-67) are observed at day 2 and want PA-12 to affect cells ability to grow on other surfaces.
day 4 (Figure 6A). The absence of p53 antigen (anti- Certain practical applications may necessitate cells to be
proliferative marker) in both cases suggests cells cultured subcultured from one type of substrate to another. Second,

Volume 9 Issue 1 (2023) 30 https://doi.org/10.18063/ijb.v9i1.623