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International Journal of Bioprinting Light-based muscle bioprinting with bioglass

2C. Figure 3A shows actual examples of the specimens myoblasts to myotubes than hydrogels with lower Young’s
fabricated using the apparatus introduced in this study for moduli (around 3 kPa).
evaluating the mechanical properties of GelMA constructs,
with or without MBGNs. Tensile assays were performed 3.3. Feasibility for printing thin features
on specimens produced both with and without MBGNs Myoblasts respond to the scale and orientation of the
(Figure 3B). The results indicated that MBGNs had no scaffold where they grow. In general, myoblasts align
significant effect on either the stress–strain curves (Figure more effectively to develop multinucleated cells when
3C) or the tensile Young’s modulus (Figure 3D). While cultured on thin features than on wide features. Therefore,
the mechanical characterization did not demonstrate an characterizing the resolution of the printer, in terms of its
enhancement effect with the addition of MBGNs to the ability to consistently fabricate thin features, is relevant
hydrogels, we found that the inclusion of the nanoparticles in the context of muscle biofabrication. 47,48 In this work,
could be facilitated by features printing and samples we fabricated GelMA constructs that exhibited features
handling. This empirical observation aligns with previous (bridges) of different thicknesses (Figure 2) to evaluate (a)
reports indicating that gelatin-based hydrogels interact the performance of our LBB system in terms of resolution
with ions released from bioactive glass nanoparticles, and fidelity, and (b) the proliferative/maturation behavior
forming ion-dipole bonds. These interactions, particularly of C2C12 cells bioprinted in features of different thicknesses
with multivalent cations (e.g., Ca ), promote hydration in GelMA constructs with or without MBGNs.
2+
and stability. 33,34 However, a more comprehensive study Representative micrographs of GelMA constructs
involving complementary fatigue and compression testing printed without and with MBGNs are presented in Figure
could provide valuable insights for implementing these 4A and B, respectively. It was observed that pristine
hydrogel formulations with tunable mechanical properties GelMA constructs tended to present some undesired
for tissue engineering applications. curvatures at the corner and exhibited lower fidelity than
For muscle bioprinting applications, an adequate constructs printed with GelMA added with MBGNs.
Young’s modulus is paramount for several reasons. Muscles This suggested that the increase of ink deformability
are dynamic tissues that undergo frequent mechanical due to the addition of nanoparticles, as measured
loading and stretching during daily activities. Hence, a during the mechanical assessment, could improve the
39
biomaterial with a relatively low stiffness (Young’s modulus printability, and enabled the fabrication of finer patterns
of approximately 12 kPa) and high flexibility is favorable (see Figure 4B). We also observed the presence of fully
to mimic the natural response to mechanical forces uncured GelMA layers on the surroundings of the
and to promote efficient myogenesis. 40,41 Furthermore, printed structures (these membranes are shown enclosed
the presence of a ductile or malleable biomaterial can with yellow frames in Figure 4C). These features can
enhance the potential for successful engraftment, reduce be observed between both GelMA constructs added
the risk of inflammation, and promote overall functional with or without nanoparticles. Typically, these traits are
recovery. 42,43 Biomaterials with these characteristics have undesired. One of the strategies for reducing this effect
been reported to favor cell proliferation and differentiation was including tartrazine (an artificial food dye that has
in vitro, as they create an environment conducive to robust been widely used in foods and pharmaceuticals for many
musculoskeletal tissue regeneration, enabling cells to years) and that has been previously used to reduce the
49
respond to the surrounding matrix, proliferate, align, and scattering of light during bioprinting. Examination of
differentiate into musculoskeletal-like tissue. The Young’s the samples using optical microscopy showed that these
modulus of skeletal muscle has been estimated to range features were more prominent in constructs printed with
from 20 to 100 kPa in various studies, considering different GelMA with MBGNs than in controls (i.e., constructs
methodologies and experimental conditions. 44,45 Notably, printed with pristine GelMA). Arguably, the refractive
the Young’s modulus of a muscle fiber changes during properties of the nanoparticles could have enhanced the
early differentiation, starting from low values (e.g., 11.5 development of these thin lateral hydrogel membranes.
± 1.3 kPa) for undifferentiated myoblasts and increasing While these features were undesired as they were not part
to values within the range reported by other authors of the original design, fluorescent microscopy showed
for mature muscle (e.g., 45.3 ± 4.0 kPa) after 8 days of that after 3–5 days these membranes sustained the
differentiation. These findings suggest that hydrogels with culture of C2C12 myoblast in cell culture experiments.
45
Young’s modulus in the range of –15 kPa may be suitable Our results suggested that the MBGNs improved C2C12
for seeding myoblasts. Within this range, a recent study myoblasts proliferation progressively increasing cellular
46
demonstrated that stiffer hydrogel matrices (approximately density in the edges of the bioprinted scaffold (see Section
7 kPa) are more effective in inducing differentiation from 3.4 for more details). Table 2 summarizes the thickness

Volume 10 Issue 4 (2024) 556 doi: 10.36922/ijb.1830