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

14 days of culture with cells, which initially adopted an These observations were further corroborated by use
expected round shape . However, as culture progressed, of qRT-PCR to assess mRNA levels of each of these three
[44]
the cells tended to migrate toward the surface where they markers. Although the immunolabeling showed promising
would form smaller clusters if there were other cells in results, the nature of some of the antibodies used, such as
the surrounding. These cell clusters were more prevalent the collagen type II polyclonal antibody, could lead to false
at day 14 of culture and resembled smaller versions of the positive results. qRT-PCR confirmed the chondrogenic
3D pellets. At this time, the cells adopted a more “spread”’ behavior observed. Firstly, although not statistically
morphology and, due to their lower numbers, lacked significant, mRNA expression levels for all three
any obvious hypoxic core or cell death. These findings chondrogenic markers were somewhat raised between day
indicate that the cells acquired an autonomous behavior 7 and 14 in the 3D pellets, supporting the use of this “gold
and attained a conformation that closely resembles a standard” system for cartilage in vitro chondrogenesis.
physiologically relevant cartilage distribution. Significant upregulation in levels of COL2, AGC, and SOX9
mRNA was observed in the hydrogel culture on day 7. This
The final investigation included assessment of supports the notion that cells respond by adopting greater
chondrogenic marker expression at both protein and chondrogenic potential in the hydrogel system during the
mRNA transcript levels. Protein expression was assessed first 7 days. The observed lack of any significant difference
by immunofluorescence using SOX-9 to determine initial in mRNA levels for these chondrogenic marker transcripts
chondrogenesis. Positive labeling for SOX-9 was seen in in the following week (day 14) suggests that cells cultured
both control and PeptiInk cultures on days 7 and 14. When in the hydrogel system exhibit an accelerated chondrogenic
chondrocytes are expanded in two-dimensional, it is known behavior.
that they tend to lose their chondrogenic phenotype after
passage 4 or 5 and behave like fibroblasts [45,46] . By assessing This hydrogel system presents promising advantages
this early chondrogenic marker, we could assess whether such as the possibility to be used as a bioink and as a human
the cells were retrieving their original phenotype after being cartilage in vitro model. The high expression of cartilage
placed in a 3D environment. Other studies have shown markers, such as collagen type II, aggrecan, and SOX-9,
[46]
that when placing previously 2D-expanded chondrocytes shows that this system also enables the chondrogenesis
[35]
into a 3D environment, they started expressing SOX-9 observed in other animal models . The significant
and later chondrogenic markers. Here, we observed high upregulation of the chosen chondrogenic mRNAs in the
intensity of intracellular nuclear SOX-9 labeling on day 7 in PeptiInk system on day 7 indicates a faster chondrogenesis
pellet cultures, which decreased at subsequent time points when placing the human primary chondrocytes in the
until day 14. SOX-9 labeling in PeptiInk culture was, by hydrogel system. However, the instability of the hydrogel
contrast, low on day 7 but instead increased in intensity by and its fast degradation rate when mixed with cells in
[44]
day 14, showing the potential induction of chondrogenic culture, which have already been reported , make it
differentiation under these conditions. difficult to maintain the culture in vitro for longer than 14
days. The loss of hydrogel volume over the culture period
Later chondrogenic markers such as collagen type II potentially impedes the further chondrogenic development
and aggrecan were also assessed. As expected, collagen of the system into mature cartilage tissue. Further research
type II and aggrecan were expressed in the cartilage pellet. should focus on the mechanism of degradation of these
More collagen type II appeared to be expressed at the hydrogels, which has already been speculated to be cell
surface of the pellet on day 7 and then increased across endocytosis . Additional strategies to slow down this
[44]
the matrix on day 14, which is an observation that differs PeptiInk degradation without disrupting the chondrogenic
from previous 3D pellet chondrocyte studies where the potential should be assessed.
production of collagen type II commences at the center
and spreads outward [40,41] . Aggrecan, on the other hand, Overall, our findings showed that the PeptiInk Alpha 1
seemed to be expressed all over the pellet from day 7 culture system does promote human chondrogenesis
onward, confirming previously reported observation . in vitro and is a suitable material for 3D bioprinting. It
[43]
The hydrogel culture showed an intracellular expression of allows for chondrocytes to survive, self-assemble and
collagen type II on day 7 and a more prominent intra- and produce chondrogenic matrix proteins and markers.
extra-cellular expression on day 14, especially in the cell This animal-free alternative has great potential to bring
cluster formations at the surface of the hydrogel. Aggrecan, research closer to an ethical and more sustainable platform
on the other hand, appeared to have a lower expression on to recreate human cartilage in vitro. Albeit further studies
day 7 which then increased in those surface cell clusters are needed to assess the feasibility of this PeptiInk to be
on day 14. used in long-term cultures for human cartilage in vitro
modeling, our novel findings showed for the first time the

Volume 9 Issue 6 (2023) 462 https://doi.org/10.36922/ijb.0899