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International Journal of Bioprinting Biomimetic scaffolds for tendon healing

and maximum stress (kPa) (Figure 5B-iv). This assay was conditions. The lower viability in the initial times could be
performed with and without cells. The results obtained related to the stress produced by the printing process.
from the force vs. displacement analysis are shown in part To analyze the metabolic activity of the different
(D) of the Supplementary File. cell densities over time, the CCK-8 reagent was used
An analysis of the initial values revealed that at day (Figure 7A). The metabolic activities of the three cell
1, the maximum stress was 76.6 ± 0.74 kPa in acellular densities increased with time. Particularly, during the
scaffolds and 64.6 ± 2.17 kPa in tissue constructs. These first 7 days, there was a more pronounced surge in cell
values coincide with those described in the literature for metabolic activity. Subsequent to this initial period,
the materials used in the ink (range between 1.5 kPa and the rate of increase in metabolic activity became more
65 kPa for collagen, fibronectin, and Gel). The other two moderate, albeit remaining positive. Among the different
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parameters are the work to break/maximum, whose value cell densities, it can be seen that there are significant
is 3.24 ± 0.37 mJ in acellular scaffolds and 2.68 ± 1.15 mJ differences between the three densities in the first three
in tissue constructs, and the tangent compression elastic sampling times, probably due to the difference in the
modulus, whose value is 145.37 ± 5.89 kPa in acellular number of cells. On both days 14 and 21, the signal became
scaffolds and 133.40 ± 24.88 kPa in tissue constructs. equal for the three cell densities. This finding suggests that
Considering the mechanical properties of the tissues, these an “equilibrium” was reached on day 14, where the limit of
values of elastic compression modulus are lower than the the number of cells in the scaffold could be observed.
ones of native tendons. However, as the objective is to To determine the effect of the printing conditions on
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regenerate partial tendon ruptures, these lower properties the cells, the amount of lactate dehydrogenase enzyme in
are more suitable. In particular, it could favor cell adhesion the culture medium (related to the amount of damaged
and proliferation, which in turn might enhance ECM or dead cells) was analyzed on days 1 and 3 after 3D
deposition. 64,65 In addition to this, it offers other benefits bioprinting (Figure S7 in the Supplementary File).
that are not usually taken into account such as the The results showed that the printing process affects the
reduction of the stress concentration (mechanical stress integrity and viability of the cells (high levels of lactate
can be symmetrically distributed), the improvement of dehydrogenase in all cell densities on day 1). The level of
the biodegradability, the promotion of the vascularization, lactate dehydrogenase in the highest cell density, i.e., 5 ×
and the reduction of the inflammatory response (generally 10 cell mL , was significantly higher than those in other
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stiffer scaffolds activate the immune system more easily), cell densities of 1 × 10 cell mL and 2 × 10 cell mL ,
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among others. 66-68 which, however, did not show significant differences
Since the first day, the mechanical parameters continued among themselves. Nevertheless, the difference between
to decrease until day 21, and in most cases, the difference the values could be explained by the different number
between days was statistically significant. This decrease was of cells in each scaffold. Lactate dehydrogenase enzyme
progressive but less pronounced compared to the change levels underwent a substantial decrease on day 3 for cell
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between day 1 and day 3. These profiles coincide with the densities at 1 × 10 cell mL and 5 × 10 cell mL . These
degradation and swelling processes. With this, and based results imply that the cells present a rapid recovery after
on the existing literature, it can be hypothesized that the the initial stress process. Likewise, they suggest that the
degradation and swelling processes are responsible, at scaffolds offer a suitable environment for cell growth
least in part, for the decrease in the mechanical properties and proliferation.
of the scaffold over time. 69,70 No significant differences 3.5. Gene expression
were observed between acellular scaffolds and tissue Gene expression analysis was used to determine the
constructs (except for the tangent elastic modulus on day expression over time of characteristic markers of tenocytes
14), although a greater variability was observed within the (Scx and Tnc), markers of synthesis of macromolecules of
tissue constructs. the ECM (Col1a1 and Col3a1), and markers of cellular
3.4. Cell incorporation activity (c-Fos and Fn1) (Figure 7B).
After characterizing the physical and mechanical properties Scx has been identified as a very important marker
of the structures, tenocytes were incorporated into the ink of tendon neoformation and cellular differentiation
to obtain tissue constructs. Cell viability was analyzed to tenocytes. It has also been reported that this gene
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qualitatively at different times and at three cell densities plays an integral role in cellular differentiation and ECM
(1 × 10 cell mL , 2 × 10 cell mL , and 5 × 10 cell mL ) organization. In this case, no significant differences in
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using the live-dead assay (Figure 6). The obtained the expression of this gene were observed over time. This
micrographs showed high cell viability in all the tested same effect has previously been reported. One possible
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Volume 10 Issue 3 (2024) 456 doi: 10.36922/ijb.2632