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International Journal of Bioprinting 3D-bioprinted peripheral nerve scaffold

calcein-AM/PI staining on days 1, 4, and 7 to assess the higher than those observed in both the PCL and hydrogel
viability of scSHEDs in the scaffolds. Live cell membranes, groups (Figure 5D and E).
labeled with calcein-AM, exhibited a green fluorescence, To further investigate the function of sciatic nerve
while dead cell nuclei, stained with PI, appeared red. The regeneration, we conducted EMG on the rats 8 weeks
majority of cells displayed green fluorescence, indicating post-experiment. The CMAPs recorded from the autograft
robust viability. The assessment of scSHED viability relied (5.50 ± 0.84 mV) and scSHED (2.05 ± 0.63 mV) groups
on the quantification of both live and dead cells; the survival exhibited significantly higher amplitudes compared to
rate was 84.98 ± 2.99% on day 1, 91.70 ± 0.42% on day 4, those of the PCL (0.90 ± 0.48 mV) and hydrogel (1.05
and 93.06 ± 2.15% on day 7 (Figure 4A and D). Notably, ± 0.17 mV) groups. NCV was also significantly higher
there was a statistically significant difference between days in both the autograft (45.72 ± 6.03 m/s) and scSHED
1 and 7 (p < 0.01). In terms of cell proliferation, our findings (42.49 ± 4.98 m/s) groups than in the PCL and hydrogel
demonstrated that scSHEDs proliferated similarly in the groups. Taken together, these results indicate enhanced
scaffold compared to previous studies, suggesting that the electrophysiological recovery in the scSHED group than in
utilization of 3D bioprinting did not adversely affect cell the PCL or hydrogel group (Figure 5F and G).
proliferation rates. This observation was consistent with
the results obtained from live/dead cell staining. An injury to the sciatic nerve can result in a loss of
The gene expression levels of S100B and BDNF were innervation in the target muscles, ultimately leading
measured in both 2D- and 3D-printed scSHEDs. It was to muscle atrophy. Eight weeks post-surgery, there was
observed that the gene expression levels were similar or higher wet muscle weight observed in both the autograft
slightly higher in the 3D scSHEDs compared to the 2D and scSHED groups compared to the PCL and hydrogel
scSHEDs (Figure 4F and G). These findings suggested that groups, albeit not statistically significant (Figure 5B and C).
3D bioprinting did not disrupt gene expression. The release Muscle morphology was assessed using Masson trichrome
of NGF from scSHEDs and scSHEDs in scaffolds was also staining. The muscle area of the scSHED group (80.91 ±
evaluated on day 7. NGF release from 2D scSHEDs and 7.33%) was significantly larger than that of the PCL and
3D scSHEDs was 50.12 ± 3.88 pg/ml and 53.90 ± 4.23 pg/ hydrogel scaffold groups (Figure 6A and D).
ml, respectively (Figure 4E), suggesting that the levels of The number of regenerated nerves was evaluated using
NGF released from scSHEDs were also not disrupted by histological analysis. Semithin toluidine blue staining of
3D bioprinting. the middle portion of the sciatic nerve in the scSHED
The protein S-100β serves as a prototypical cytoplasmic group revealed a higher abundance of well-myelinated
marker protein for Schwann cells, playing a crucial role in axons compared to those in the PCL and hydrogel groups.
nerve cell proliferation and functional protein expression. There were significantly more myelinated nerve fibers
2
As displayed in Figure 4B and H, most cells exhibited per 10,000 μm in the sciatic nerve of the scSHED group
robust S-100β expression, indicating that neither the (107.0 ± 4.0) than in the PCL (87.0 ± 7.0) and hydrogel
3D bioprinting process nor the 3D culture impeded (74.3 ± 3.79) groups. Similarly, myelinated nerve fibers had
its expression. Cytoskeletal staining elucidated the a larger diameter in the scSHED group (7.97 ± 3.22 μm)
morphological characteristics of the printed cells, which compared to both the PCL (6.24 ± 1.70 μm) and hydrogel
were strongly stretched in a spindle pattern adhered to the (6.28 ± 1.46 μm) groups (Figure 6B, C, and E).
3D structure. This observation aligned with the findings The expression levels of GAP43, NF200, and S-100β
from the S-100β analysis; however, no distinct polarity was were assessed using immunohistochemistry in the four
observed (Figure 4I). groups. We observed significant increases in the expressions
of GAP43, NF200, and S-100β in both the autograft and
3.4. In vivo analysis of 3D-printed scSHEDs scSHED groups compared to the PCL or hydrogel group.
We used gait analysis to assess the restoration of sciatic The differences in GAP43, NF200, and S-100β expressions
nerve function after the experiment. In general, a decline in between the scSHED group and both the PCL and hydrogel
sciatic nerve function leads to narrowing of toe extension groups were statistically significant (Figure 7).
and intermediate toe extension. Eight weeks after surgery,
there was a significant improvement in lameness among 4. Discussion
rats, in both the autograft and scSHED groups. Footprint
analysis demonstrated that the autograft and scSHED SHEDs have a higher proliferation rate than DPSCs and
groups exhibited greater toe extension and intermediate can be readily obtained from deciduous teeth or discarded
toe extension compared to the PCL and hydrogel groups. without any ethical concerns. 45,46 Generally, SHEDs can
Notably, the SFIs in the scSHED group (-68.07 ± 6.91) were stimulate capillary angiogenesis and differentiate into

Volume 10 Issue 4 (2024) 468 doi: 10.36922/ijb.2908