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International Journal of Bioprinting Light-based muscle bioprinting with bioglass

probably attributed to the high cell density observed in differentiation of myoblasts to myofibrils during extended
these samples, leading to nutrient and oxygen deprivation, culture times. For this, GelMA C2C12-laden constructs
which in turn resulted in cell death. In a previous report, bioprinted with and without MBGNs were stained with
we used the same type of MBGNs in similar concentrations actin/DAPI for examining the alignment and elongation
to foster the proliferation and alignment of C2C12 cells of the myoblasts in the absence and presence of the
in alginate/GelMA hydrogel fibers. In that experiment, nanoparticles (Figure 6A). We observed cell elongation
32
we also observed that C2C12 cells attached and spread in and development of multinucleated cells in both GelMA
GelMA constructs during the first 12 h of post-bioprinting constructs with and without MBGNs. We also observed
culture in both pristine GelMA and GelMA with MBGNs clear patterns of cell alignment, a characteristic highly
constructs. The usage of conventional bioprinting sought after for muscle tissue engineering, in both the
strategies (i.e., extrusion and inkjet) often necessitates very construct enriched and not enriched with nanoparticles.
long time (more than 24 h) for monitoring cell attachment In this study, the degree of alignment was determined by
and elongation in 3D hydrogel environments (frequently image analysis, and reported as the percentage of elongated
alginate/GelMA mixes). Attachment and elongation are cells aligned within a range of 30° with respect to the
8
processes commonly performed by anchorage-dependent direction of the edge of the construct (Figure 6B).
cells and influenced by the hydrogel composition and Over time, both types of constructs showed an increase
stiffness, and the printing strategy. Here, by using this in cell alignment. Interestingly, cell alignment was not
cost-effective laser-based printer, we were able to fabricate homogeneous throughout the different sections of the
cell-laden constructs in which C2C12 myoblasts rapidly constructs (Figure 6C and D); of the bioprinted structures,
attached and elongated in GelMA hydrogels. GelMA is significantly higher levels of alignment were observed
widely used in tissue engineering applications due to its at construct edges than at the internal (i.e., centermost)
well-appreciated properties for fostering cell attachment, section. In addition, a higher degree of alignment was
by virtue of the presence of arginine-glycyl-aspartic acid observed in constructs added with MBGNs than in
(RGD) motifs and metalloprotease sites that facilitate matrix constructs without nanoparticles. For example, nearly 70%
remodeling. The bioprinter used in this work enables of the cells located in the internal section of the constructs
22
the use of GelMA hydrogels, instead of GelMA/alginate added with MBGNs exhibited alignment on day 15. In
mixes, in the fabrication of 3D muscle-like microtissues. contrast, 80% of cells were aligned to the edges in the
We believe that this will contribute to expanding the range border of the constructs. Furthermore, the cell alignment
of printing applications by making possible the effective in the constructs without MBGNs was 42% at the internal
crosslinking of GelMA constructs without the need for the section and 75% at the external edges. Notably, even from
use of alginate and calcium chloride solutions. Moreover, day 1, cells exposed to MBGNs exhibited higher levels of
our results showed that, in the range of printing conditions alignment compared to the control cells. Results suggest
used in this study in terms of exposure time and light that the nanoparticles promote cellular alignment and
intensity, the cell viability was not compromised. foster a favorable microenvironment for muscle tissue
We also evaluated the metabolic activity using in C2C12- development. Besides, our results also demonstrated that
laden constructs during the first 15 days of culture. Presto adding MBGNs nanoparticles to the GelMA constructs
Blue assays indicated an increase in cellular metabolic could enhance metabolic activity and total cell count (as
activity over the first 9 days of culture (Figure 5C), and determined by nuclei counting). The number of nuclei per
no significant differences were observed between the unit of area was significantly higher in GelMA constructs
metabolic activity determined in GelMA constructs with enriched with MBGNs than in pristine GelMA constructs
or without nanoparticles during this period. However, we (Figure 6E). These observations could be attributed to the
observed a higher metabolic activity in GelMA constructs release of Si and Ca from MBGNs, which may enhance
2+
4+
enriched with MBGNs than in pristine GelMA constructs cellular metabolism and myogenic differentiation. 28,29
on day 15 of culture. These results suggest that MBGNs However, further exploration of cellular markers of
are not only cytocompatible but may also be related myogenesis could provide valuable insights into the
to a dynamic cellular process associated to a stage of underlying mechanisms driving these observations.
differentiation (e.g., from myoblasts to myocytes, or from Indeed, the application of bioactive glasses in muscle tissue
myocytes to myotubes). This finding is consistent with repair is receiving increasing attention. 28,29
previous reports and is supported by the cell-oriented
morphology observed in the constructs at this time point. 32 4. Conclusion
Next, we analyzed the effect of the bioprinting process This work evaluated the performance of an affordable LBB
and the addition of MBGNs in the elongation and system, retrofitted from a commercial SLA 3D-printer, for

Volume 10 Issue 4 (2024) 559 doi: 10.36922/ijb.1830