Page 259 - IJB-10-6

International Journal of Bioprinting 3D-printed contractive pennate muscle

the bioink. The extruder was connected to an air pump, h rest), the tissues were incubated in DM for 7 days, during
which extruded the bioink at the appropriate air pressure. which the culture medium was changed daily. We then
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Simultaneously, it followed the predetermined route in studied the differentiation of myoblasts into myotubes using
the slicing software, printing the muscle tissues layer-by- fluorescence staining of F-actin and nuclei and subsequently
layer in the support mold (Figure 5C). After printing, the characterized the differentiation level of C2C12 cells via
3D-printed pieces were crosslinked under 405 nm light confocal laser scanning microscopy (CLSM).
using a portable curing light source (LS-1601; EFL, China),
and the supports were removed. 2.7. Myotube alignment analysis in
engineered tissues
The quality of muscle tissue morphology includes Myotube alignment of skeletal muscle cells was analyzed
macroscopic shape dimensions and microscopic structure using fluorescence staining and subsequent observation
dimensions. For macro dimensions, the length and major under a laser confocal microscope (A1; Nikon, Japan).
diameter of the tissue were measured using a micrometer After a seven-day culture, the 3D-printed pieces were
and thickness gauge, respectively. For the microstructure cut into thin slices along the long axis, washed, fixed, and
dimensions, a layer containing microchannels from the permeabilized. The samples were incubated in Alexa Fluor
sliced model of tissue was printed separately, and the 488-conjugated phalloidin (1:200) at room temperature in
width of the microchannels was then measured under a the dark for 30 min and rinsed with PBS after incubation.
stereomicroscope (SMZ745; Nikon, Japan). The cell nuclei were then stained with a DAPI staining

2.5. Cell viability evaluation solution for 30 s (the detailed staining procedure is
Cell viability was analyzed on days 1, 3, 5, and 7 after 3D described in the Supporting Information). A laser confocal
printing using fluorescent labeling with molecular probes microscope was used for confocal imaging of skeletal
from the Live/Dead Viability/Cytotoxicity Kit. Since the muscle cell nuclei to assess myotube alignment. ImageJ
printed muscles were not exposed to electrical stimulation software was used to identify the long-axis angle of each cell
to induce cell differentiation before cell viability evaluation, nucleus. Origin software was used to conduct a frequency
the cells retained their ability to proliferate. The culture distribution analysis of the angular orientation, calculating
medium was changed daily during cultivation to remove the frequency of cell orientations in each 20° interval from
dead cells from the muscle. Molecular probes were used −95° to 85°. Cells with an angle <30° between the long axis
to characterize cell activity and observe cell growth in of the nucleus and the orientation direction were assumed
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the 3D-printed muscle tissues. The staining solution was to be oriented cells. Additionally, we investigated the
prepared under dark conditions by adding 1 mL PBS to effect of 3D printing parameters on cell orientation rate,
an Eppendorf tube, followed by 2 μL EthD-1 and 0.5 μL and various printing speeds (6, 8, and 10 mm/s) and air
calcein AM. The tissues were washed with PBS once and pressure (2, 3, and 4 kPa) were considered in this study.
incubated in the mixed staining solution in the dark for 2.9. Contractility evaluation
15 min. The tissues were washed with PBS three times and Contraction displacement of tissues was measured
observed under an inverted fluorescence microscope (Ti-S; without load under the electric field, while contraction
Nikon, Japan). Stained live cells emitted green fluorescence force was measured when loaded with U-shape posts
(Ex/Em = 488/500 nm) under blue light excitation, while made of PDMS. Electrical stimuli were applied to induce
dead cells emitted red fluorescence (Ex/Em = 560/600 nm)
under green light excitation. ImageJ software was used to contraction and displacement. The electrical signal
count the number of living and dead cells. The cell survival generator was used to generate a pulse square wave with
rate was calculated as the ratio of the number of living cells adjustable amplitude and frequency (1, 2, and 4 Hz).
to the total number of cells. The signal generator was connected to a booster device
to create an electric field between the two electrodes (1,
2.6. Cell differentiation with electrical stimulation 2, and 4 V/mm). A microscope thermo plate (TP-C-108;
The 3D-printed tissues were cultivated with 5 mL DM Mshot, China) was utilized to maintain the media at 37°C,
after incubating in GM for 3 days. After 24 h cultivation providing an appropriate temperature for the muscle
in DM, electrical field stimulation was generated by a tissues. A stereomicroscope (SMZ745; Nikon, Japan) was
signal stimulator (DG812; Rigol, China) as square waves used to observe the contraction process and record videos;
(amplitude: 1.0 V/mm; pulse duration: 10 ms; frequency: 1 the load-free contraction displacement was measured
Hz) and transmitted to platinum electrodes fixed on both using Tracker software; the displacement of posts was
sides of the petri dish. After electrical field stimulation measured to compute the force exerted against the posts
was performed on the tissues with the corresponding after electrical stimulation using Euler-Bernoulli’s beam
parameters for 10 h (every 1 h stimulation followed with 1 bending equation:

Volume 10 Issue 6 (2024) 251 doi: 10.36922/ijb.4371