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. 2024 Dec 23;19(12):e0310711.
doi: 10.1371/journal.pone.0310711. eCollection 2024.

Nerve regeneration using a Bio 3D conduit derived from umbilical cord-Derived mesenchymal stem cells in a rat sciatic nerve defect model

Terunobu Iwai  1 , Ryosuke Ikeguchi  1   2 , Tomoki Aoyama  3 , Takashi Noguchi  1 , Koichi Yoshimoto  1 , Daichi Sakamoto  1 , Kazuaki Fujita  1 , Yudai Miyazaki  4 , Shizuka Akieda  4 , Tokiko Nagamura-Inoue  5 , Fumitaka Nagamura  6 , Koichi Nakayama  7 , Shuichi Matsuda  1
Affiliations

Nerve regeneration using a Bio 3D conduit derived from umbilical cord-Derived mesenchymal stem cells in a rat sciatic nerve defect model

Terunobu Iwai et al. PLoS One. .

Abstract

Human umbilical cord-derived mesenchymal stromal cells (UC-MSCs), which can be prepared in advance and are presumed to be advantageous for nerve regeneration, have potential as a cell source for Bio 3D conduits. The purpose of this study was to evaluate the nerve regeneration ability of Bio 3D conduits made from UC-MSCs using a rat sciatic nerve defect model.

Methods: A Bio 3D conduit was fabricated using a Bio 3D printer by placing UC-MSC spheroids into thin needles according to predesigned 3D data. The conduit was transplanted to bridge the 5-mm gaps of Lewis rat sciatic nerve, and nerve regeneration was evaluated at 8 weeks (Bio 3D group). Transplantation of autologous nerve segments (autograft) and silicone tubes represented the positive and negative control groups, respectively. In a second experiment, immunological reactions were evaluated in Bio 3D, autograft, and allograft groups by histochemical staining of transplanted segments in Brown Norway rats.

Results: The mean angle of attack value in the kinematic analysis was significantly better in the Bio 3D group (‒20.1 ± 0.5°) than in the silicone group (‒33.7 ± 1.5°) 8 weeks after surgery. The average diameters of myelinated axons were significantly larger in the Bio 3D group (3.61 ± 0.15 μm) than in the silicone group (3.07 ± 0.12 μm), and the number of myelinated axons was significantly higher in the Bio 3D group (11,201 ± 980) than in the silicone group (8117 ± 646). Histological findings (hematoxylin and eosin [HE] staining and anti-CD3 fluorescent immunostaining) showed that rejection was suppressed in the Bio 3D group compared to the allograft group. Based on macroscopic findings and histological findings (anti-human mitochondrial fluorescent immunostaining), UC-MSCs in the Bio 3D conduit disappeared gradually from week 1 to week 8.

Conclusions: The Bio 3D conduit prepared from UC-MSCs was superior to the silicone tube and achieved comparable nerve regeneration to the autologous (autograft) group. Rejection was suppressed in the Bio 3D group compared to the allograft group. Although this study used a xenograft model, we speculate that rejection was low due to the characteristics of UC-MSCs. UC-MSCs are a useful cell source for Bio 3D conduits.

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Conflict of interest statement

There are no patents or marketed products to declare. KN is the co-founder and shareholder of Cyfuse Biomedical K.K., Tokyo, Japan (Cyfuse). YM and SA, who are employees of Cyfuse, contributed to the manufacturing of 3D conduits and Cyfuse provided the bioprinter to manufacture the conduit. The company has the industrial rights related to the bioprinting method used to construct the 3D conduit in this work. Cyfuse provided support in the form of salaries for authors YM, SA and KN and provided research grants to TA, KN and SM. These competing interests do not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1
Three types of materials were transplanted into the 5-mm gaps of rat sciatic nerve: (A) Bio 3D conduit, (B) autograft conduit, and (C) silicone conduit. Regenerated nerves 8 weeks after surgery in the (D) Bio 3D group, (E) autograft group, and (F) silicone group. Nerves were regenerated in all three groups, but the regenerated nerves in the silicone group were thinner than those in the Bio 3D and autograft groups.
Fig 2
Fig 2
(A) Angle of attack (AoA) is formed by the toe and metatarsal bone immediately before the toe touches the ground at the end of the swing phase. (B) For DT, there was no significant difference among the three groups. (C) For AoA, the Bio 3D conduit performed significantly better than the autograft and silicone conduits. (D) The blue waveform is stimulated from the greater trochanter, and the green waveform is stimulated from the popliteal fossa (vertical frame: 1 ms, horizontal frame: 2 mV). (E) CMAP, and (F) NCV. For CMAP and NCV, the autograft was significantly better than the silicone conduit, and there was no significant difference between the Bio 3D conduit and the autograft. DT: Drag toe, AoA: Angle of attack, CMAP: Compound motor action potential, NCV: Nerve conduction velocity. Error bars represent the standard error.
Fig 3
Fig 3
The tibialis anterior muscle on the right side is the healthy side, and the muscle on the left is the transplanted side: (A) Bio 3D group, (B) autograft group, (C) silicone group. (D) Based on the wet weight of the tibialis anterior muscle of the transplanted side, the autograft group showed significantly better recovery than the silicone group. Error bars represent the standard error.
Fig 4
Fig 4
(A-C) Semi-thin transverse sections stained with toluidine blue (scale bar = 1000 μm): (A) Bio 3D group, (B) autograft group, (C) silicone group. (D-F) Ultrathin transverse sections observed with transmission electron microscopy (scale bar = 2 μm): (D) Bio 3D group, (E) autograft group, (F) silicone group. The regenerated nerves of the Bio 3D group were larger than those of the silicone group and similar to those of the autograft nerve group. (G) Number of myelinated axons, (H) diameter of myelinated axons, and (I) myelin sheath thickness. Significant differences were observed between the Bio 3D group and the silicone group, and between the autograft group and the silicone group in all three parameters. Error bars represent the standard error.
Fig 5
Fig 5. Regenerated nerve 1 week after surgery, illustrating nerve rejection.
(A-C) HE staining of the transverse section in each of the three groups (scale bar = 50 μm). The infiltrative mononuclear cells are indicated by white arrows. We counted infiltrative mononuclear cells in the nerves and evaluated the inflammatory response. (D) Number of mononuclear cells infiltrating the nerves within the view field on HE staining. There were significantly lower counts in the Bio 3D group and the autograft group than in the allograft group. Error bars represent the standard error. (E-G) Anti-CD3 antibody staining of the transverse section in each of the three groups (scale bar = 500 μm). We considered the area indicated by the arrow below to be infiltrative mononuclear. (H-J) Anti-CD3 antibody and DAPI staining of the transverse section in each of the three groups (scale bar = 500 μm). Perineural staining in the Bio 3D group and the allograft group was observed as staining around the edge of the nerve. (K) Number of bright spots of anti-CD3 antibody. There were significantly lower counts in the Bio 3D group and the autograft group than in the allograft group. Error bars represent the standard error.
Fig 6
Fig 6. Immunohistochemical staining of the regenerated nerve 8 weeks after surgery showing nerve recovery.
RNF402 is stained green, S100 is stained red, and the nucleus is stained blue using DAPI (A-C). The upper row shows merged images (RNF402/S100/DAPI) of the transverse section in each of the three groups (scale bar = 500 μm). (D-F) The middle row shows images of the transverse section in the Bio 3D group (scale bar = 50 μm). (G-I) The lower row shows images of the longitudinal section in the Bio 3D group (scale bar = 50 μm). Color development of RNF402 and S100 suggests the presence of nerve tissue.
Fig 7
Fig 7. Immunohistochemical staining to evaluate the change in the transplanted bio 3D conduit within the rat were between 1 week and 8 weeks after surgery.
(A-C) Anti-mitochondria antibody staining of the transverse section in each of the three groups (scale bar = 500 μm): (A) week 1, Bio 3D group, (B) week 8, Bio 3D group, (C) week 1, autograft group. (D) There were significantly greater values in the week 1 Bio 3D group than in the week 8 Bio 3D group and the week 1 autologous group. Error bars represent the standard error.
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