Page 260 - IJB-10-6

International Journal of Bioprinting 3D-printed contractive pennate muscle

a
F = 3 EI γ () (I) pennate muscle model provides greater contraction force
z
a 3 compared to the parallel muscle model under the same
deformation, demonstrating the superior properties of the
where F is the contraction force, E is the Young’s pennate muscle structure.
modulus, I is the second moment of area of the post 3.2. Morphology analysis of the engineered
Z
around the z-axis, a is the height at which tissue is pulling muscle tissues
from the post, and γ(a) is the displacement of the post at The macroscopic shape of the 3D-printed muscle tissues is
that height. displayed in Figure 5D, and its dimension was measured

2.10. Statistical analysis and analyzed accordingly (Figure 5E); the long axis was
All experimental results were processed and plotted using 12.18 ± 0.06 mm, and the major diameter was 4.37 ±
Origin software (OriginPro 2021; OriginLab Corporation, 0.03 mm (n = 6). Compared to the designed dimensions
USA). The data are expressed as mean ± standard in Figure 4, the fabrication error of the long axis is about
deviation. Statistical analysis was performed via one-way 1.52%, while that of the major diameter reaches 9.33%.
analysis of variance (ANOVA), and p < 0.05 was considered To accurately measure the width of the microchannels
statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001). in the engineered tissues, we printed a separate layer of
microchannels from the layer-by-layer printing process.
3. Results Figure 6A displays the microchannel size in the muscle
tissues observed under an inverted microscope; the average
3.1. Structural design of the engineered tissue after measured width was 284.28 ± 14.40 μm (n = 3). There
optimization was no significant difference between the measured and
The in vitro skeletal muscle in our study should possess designed values of the width of the printed microchannels
macro and microstructures similar to the gastrocnemius (Figure 6B), indicating that they meet the requirements for
muscle of frogs. Based on our observations and nutrient transport and cell survival.
measurements of the gastrocnemius muscle, as well as
our production capability, specific tissue dimensions were 3.3. Cell status analysis
selected (i.e., L: 12 mm, R: 4 mm, r: 2 mm, and pennate Live/dead staining was performed on sliced tissues on
angle: 15°; Figure 3). Simultaneously, we conducted days 1, 3, 5, and 7 after printing. Figure 6C demonstrates a
simulation experiments on models with different macro steady increase in cell viability over time, reaching 79.89%
and microparameters, demonstrating the rationality of on day 7 after printing. Under electrical stimulation in
our parameter selection (Figures S1 and S2, Supporting DM, the skeletal muscle cells would differentiate into
Information). The simulation results on the deformation muscle fibers that are essential for the functionalization of
distribution of the model revealed that maximum muscle tissues. Myotube alignment was compared between
deformation occurred at the end face, where the contractile the pennate and parallel muscle tissues under different
force was exerted (Figure 4B). This phenomenon is printing parameters (printing speed: 6, 8, and 10 mm/s;
consistent with the characteristics of compression air pressure: 2, 3, and 4 kPa). The differentiation status
deformation. However, in the actual contraction process of was observed via fluorescence staining of the cytoskeleton
the muscle tissues, the deformation distribution should be (Figure 6D), and the angle of the nucleus was calculated to
more uniform than the simulation results, as the muscle assess the degree of cell alignment. Figure 6E displays the
fibers are evenly distributed inside. frequency distribution of cell nucleus angles (S: printing
speed [mm/s]; P: air pressure [kPa]; cell orientation rate:
Furthermore, simulations were conducted to compare S6P2: 44.74%, S8P2: 45.87%, S10P2: 51.93%, S6P3: 41.04%,
the deformation performance of the designed pennate S6P4: 45.22%). We noticed that the cell orientation rate
tissue model with that of the parallel muscle model, both increased with higher printing speed, while no significant
with the same macro geometry. In Figure 4B, results correlation was established between air pressure and cell
indicate no significant difference in the deformation orientation rate. The highest orientation rate was 51.93%,
displacement between the two muscle configurations obtained at a printing speed of 10 mm/s and an air pressure
under the same contraction force. The simulation results of 2 kPa.
displayed an average deformation strain of 0.1308 for
the pennate muscle. In contrast, the average strain of the 3.4. Contraction performance of muscle tissues
parallel muscle model was 0.1334. As a result, the pennate Contraction performance was evaluated based on the
muscle model exhibited an average deformation that contraction displacement and force. The test platform was
was 1.95% lower compared to the parallel muscle model. constructed (Figure 7A), and the contraction displacement
Based on the simulation results, it can be inferred that the was first measured without any load. The configuration of

Volume 10 Issue 6 (2024) 252 doi: 10.36922/ijb.4371