Page 464 - IJB-9-6

International Journal of Bioprinting 3D bioprinting of in vitro cartilage tissue model

two selected pressures. Comparison to theoretical filament across the bioprinted Alpha 1 hydrogel constructs on day 0.
width, which corresponds to an internal nozzle diameter Cell distribution homogeneity demonstrated efficiency in
(250 μm, 25G), was also performed. This showed a high the bioprinting process. We observed that the cells adopted
shape fidelity and printing consistency, with a low standard a typical chondrogenic rounded morphology. Migration of
deviation and high reproducibility (Figure 1E). Although the cells toward the surface of the Alpha 1 hydrogel was
higher printing speeds approached the theoretical filament observed over the culture time, with cells forming clusters,
width when printed at 8 kPa, a more conservative printing which became even more prominent by day 14 (Figure 3).
speed was chosen to develop the targeted structures to avoid In these cell clusters, a change in the cell morphology
printing inconsistencies. A pressure range of 8–10 kPa and from rounded to more spread out can be seen. A higher
a printing speed of 5 mm/s were selected when printing eosin staining intensity was observed within these clusters,
with the 25G conical nozzle. A compact cylindrical shape showing a higher level of ECM secretion. Single cells that
structure was chosen to 3D-bioprint the in vitro cartilage are distributed across the gel and not in the cell pellets
tissue models (Figure 1F). These models had a 5 mm retained their rounded morphology and had low levels of
diameter, 1 mm thickness, and 60% infill density. ECM around them. The 3D pellet showed a circular cell
morphology and had high levels of eosin staining in the
3.2. Establishing cellular viability post-printing in inter-cellular spaces as seen in Figure 3, indicating the
short- and long-term culture presence of ECM. Across the two time points observed,
Viability of cells in constructs was assessed post-printing there was a decrease in number of nuclei observed at the
with calcein-AM (alive) and ethidium homodimer (dead). center of the pellet, suggesting some level of cell death
Post-printing cell viability and material cytocompatibility at the core of the cell cluster, and further confirming the
were studied to evaluate the effect of the extrusion process previously reported behavior in 3D pellets .
[37]
on cell viability. For this, human primary chondrocytes
were encapsulated in the PeptiInk and bioprinted. Cell 3.4. Cartilage-specific protein marker analysis
viability was assessed 2 h after extrusion and on days 7 Chondrocyte differentiation was assessed by
and 14 of culture, using cell pellet cultures as the control. immunofluorescence for specific markers. Labeling for
Pellet cultures showed 100% viability at day 0, 2 h after the early chondrogenic marker, SOX-9, was performed to
centrifugation, which was significantly decreased (p < investigate whether primary cells adopted a chondrogenic
0.0001) to 46% at day 7 (Figure 2A) with a high number of phenotype. It revealed positive labeling in cell nuclei in
dead cells in the pellet core, and similar viability (54%) was both the 3D pellet and the Alpha 1 systems on days 7 and
found at day 14. The cell death observed in the 3D pellet 14. The intensity of the SOX-9 labeling was increased on
culture was expected as it has been previously reported day 14 in Alpha 1 in comparison to day 7. In contrast, the
that hypoxic conditions lead to a necrotic core . In intensity decreased in the cell pellet culture (Figure 4). An
[37]
contrast, cells in Alpha 1 PeptiInk showed a 30% viability increase of SOX-9 over 14 days of culture was expected, and
post-printing, with significant increases to 59% by day 7 it has been previously reported that the SOX-9 expression
and remaining stable (59%) thereafter until day 14. Cell increased in chondrocytes in the first 7 days of 3D culture
number changes assessed by DNA quantification showed post 2D expansion . Here, we assessed the expression
[38]
that the 3D pellet control exhibited a high starting DNA beyond the 7 days to explore if the increase of expression
quantity (58.5 ng/mL), which significantly decreased over was maintained at later time points; this increase in SOX-
the 14 days (by 75%, Figure 2B). Behavior of cells in Alpha 9 expression and maintenance in the PeptiInk culture
1 differed, with significant decreases in DNA content is important to discriminate between dedifferentiation
(40%) over the first 7 days but only a further 10%, non- of embedded chondrocytes and potential osteoblast
significant decrease by day 14, confirming that DNA levels redifferentiation processes . Decreases in SOX-9
[39]
were maintained during the second week of culture in expression in the 3D pellet culture show that chondrogenic
Alpha 1, which corresponds to the cell viability previously behavior observed in vivo is not evident. Negative
[39]
reported [29,31,35] .
controls can be found in Figure S2 (Supplementary File).
3.3. Extracellular matrix formation, cell morphology, Later chondrogenic markers, collagen type II and
and cell distribution analysis aggrecan, were also assessed by immunochemistry. The
H&E staining used to assess extracellular matrix (ECM) 3D pellet control showed an increase in collagen type II
production, cell morphology, and distribution shows expression from day 7 to day 14, appearing firstly at the
homogeneous cell distribution and circular cell shape surface of the pellet and then expanding all around the

Volume 9 Issue 6 (2023) 456 https://doi.org/10.36922/ijb.0899