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International Journal of Bioprinting Light-based muscle bioprinting with bioglass

of 1.2 mm (Figure 2C). Subsequently, the testing constructs 200 kDa. A lyophilization process was performed on the
were printed using the same printing parameters used in sample for 5 days and then stored at −80°C until use. For
the bioprinting processes and rinsed in phosphate-buffered the experimental study, batches of GelMA with 7.5% and
saline solution (PBS) for 24 h. During the testing process, 10% concentrations were mixed with LAP (0.1% w/v) for
a crosshead speed of 10 mm/min was maintained with photoinitiation. Since LAP is photosensitive, care was taken
the assistance of a 5 N load cell. Each experimental group to handle the material in the dark. Tartrazine was used in
consisted of four samples. The load at the point of rupture a concentration of 0.005% w/v to prevent light scattering.
was recorded, and the stress was determined by dividing For the GelMA and MBGNs bioink, a concentration of
the load by the construct cross-sectional surface area. The 0.5% of MBGNs was poured and sonicated in half of the
Young’s modulus was determined by calculating the slope DPBS solution to prevent GelMA denaturalization. The
of the initial linear region of the stress–strain curve. concentration of MBGNs was selected considering previous
literature that reported values below 1%. 37,38 Furthermore,
2.5. Pattern scaffold feature assessment it has been reported that myoblasts exhibit high metabolic
We evaluated the resolution—the printer ability to activities that provide favorable environments within these
fabricate constructs with fine/thin features—by comparing concentration values. The solution was later mixed and
32
the thickness of the features of constructs printed with sterilized via filtration using a 0.22 µm filter. C2C12 cells
the geometry shown in Figure 2A versus those of the were extracted from the culture flask and centrifuged to
corresponding STL model (ideal dimensions). The selected form a cell pellet for cell seeding. The supernatant media
geometry exhibits thin features (i.e., bridges or filaments) was removed, GelMA solution was added to the cell pellet
within the range of 35–280 µm (see Table 1). The values and resuspended by gentle pipetting to form a homogenous
were selected considering pixel size of the LCD screen cell suspension (4 × 10 cells/mL). Finally, the bioink was
6
of the apparatus. This evaluation of the resolution was loaded into the vat for bioprinting.
conducted using 10% GelMA without MBGNs (control)
and 10% GelMA enriched with 0.5% MBGNs. The 2.7. Cell culture
experiments were conducted with two replicas. The length C2C12 myoblasts (CRL-1772) were acquired from the
of the features W to W was measured and averaged American Tissue Culture Collection (ATCC, Manassas,
h
a
at the middle and the beginning, and an Axio Observer VA, USA). Briefly, early-passage cells were cultured in a
z1 microscope was used to measure the thickness of the T75 cell culture flask (Corning Inc., Corning, NY, USA)
features (W to W ) at three different regions of the fiber. with Dulbecco’s Modified Eagle Medium (DMEM; Sigma-
h
a
The absolute mean deviational error for each feature was Aldrich, St. Louis, MO, USA) supplemented with a 10%
evaluated using Equation I. fetal bovine serum (Gibco, ThermoFisher, Waltham,
MA, USA) and 1% antibiotic-antimycotic and penicillin-
streptomycin (ThermoFisher, Waltham, MA, USA)
Nominal − Experimental
MAE feature = Nominal ×100 (I) under a humidified incubator (Sanyo, Osaka, JP) at 37
°C and 5% CO . After the cells reached 75% confluence,
2
subcultures were made using 0.05% trypsin-EDTA (Gibco,
Where MAE feature denotes the mean absolute error of ThermoFisher, Waltham, MA, USA) for 5 min.
the feature, Nominal is the length of the intended feature
found on the CAD file AND Experimental is the actual 2.8. Cell viability and morphology
value measured experimentally using microscopy. For cell viability, we evaluated cell-laden C2C12 constructs
at different experimental times using Live/Dead reagent
2.6. Bioink preparation (Sigma-Aldrich, USA) according to manufacturer
Type A porcine skin gelatin, methacrylic anhydride (MA), instructions. Constructs were placed on petri dishes
Dulbecco’s phosphate-buffered saline (DPBS), and lithium washed with 1× PBS and soaked with Live/Dead working
phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) were solution for 30 min. Afterwards, Live/Dead solution was
purchased from Sigma Aldrich (Sigma Aldrich, MO, USA). removed, and constructs were washed with 1× PBS. For
The type A porcine skin gelatin was dissolved in DPBS at cell morphology, we used a Zeiss inverted microscope
60°C with a 10% w/v concentration. After homogenization, coupled with an apotome system and colibri light panels.
MA was added with a syringe pump at a flow rate of 0.5 We analyzed cell morphology using bright fields, and
mL/s at a concentration of 10% w/v to add the methacrylate live and dead cells were detected using FITC (excitation/
substitution groups. To stop the reaction, the mixture was emission 488/515 nm) and Texas Red (excitation/
diluted with DPBS four times the total volume. A dialysis emission 570/602 nm) filters. For actin filaments and
process of at least 5 days was performed at 40°C using a nuclei evaluation, actin/DAPI staining was performed
dialysis tubing cellulose membrane with a pore size of using 647 Phalloidin (Abcam, USA) and DAPI (Thermo-

Volume 10 Issue 4 (2024) 554 doi: 10.36922/ijb.1830