Effect of Pre-Induced Mesenchymal Stem Cell-Coated Cellulose/Collagen Nanofibrous Nerve Conduit on Regeneration of Transected Facial Nerve

Abstract

(1) Objective: In order to evaluate the effect of a pre-induced mesenchymal stem cell (MSC)-coated cellulose/collagen nanofibrous nerve conduit on facial nerve regeneration in a rat model both in vitro and in vivo. (2) Methods: After fabrication of the cellulose/collagen nanofibrous conduit, its lumen was coated with either MSCs or pre-induced MSCs. The nerve conduit was then applied to the defective main trunk of the facial nerve. Rats were randomly divided into three treatment groups (n = 10 in each): cellulose/collagen nanofiber (control group), cellulose/collagen nanofiber/MSCs (group I), and cellulose/collagen nanofiber/pre-induced MSCs (group II). (3) Results Fibrillation of the vibrissae of each group was observed, and action potential threshold was compared 8 weeks post-surgery. Histopathological changes were also observed. Groups I and II showed better recovery of vibrissa fibrillation than the control group. (4) Conclusions: Group II, treated with the pre-induced MSC-coated cellulose/collagen nanofibrous nerve conduit, showed the highest degree of recovery based on functional and histological evaluations.

Keywords: cellulose; collagen; nanofibrous nerve conduit; nerve regeneration; pre-induced mesenchymal stem cell.

Figure 1

The expression of specific markers was identified by real-time PCR. Transcripts of neuro-progenitor markers (CD133, GFAP, Musashi, and Nestin), trophic factors (ANG, BDNF, and VEGF), and p21 were significantly increased in pre-induced-MSCs (t-test, * p < 0.05, ** p < 0.005, mean ± SD n = 6). Control-MSCs: control-MSCs. PreIn-MSCs: preinduced-MSCs which are incubated with pre-induction media for 24 h.

Figure 2

Control (Left panel) and pre-induced MSCs (Right panel) were fixed and stained with specific antibodies, Muisashi, Nestin, ANG, GFAP, and p21, and visualized under fluorescent microscopy (red and green). Blue indicates nucleus. Pre-induced MSCs indicates preinduced-MSCs which are incubated with preinduction media for 24 h.

Figure 3

Scanning electron microscopic view of control group and MSC loaded group. Both groups show cellulose/collagen nanofibrous structure (2.0K). The magnified view shows well coated MSCs to the nanofibrous strand (arrow indicates).

Figure 4

Recovery of vibrissae fibrillation is more pronounced in groups I and II than in the control group. The highest degree of recovery of vibrissae fibrillation is observed in group II (pre-induced MSC). Repeat measure ANOVA, Between time interval; p = 0.0226, Between each group; p = 0.0047. Values of * p < 0.05, *** p < 0.001 were considered statistically significant.

Figure 5

All groups show well-regenerated nerve. Compared with the control group, both the mesenchymal stem cell (MSC) and pre-induced MSC groups show slightly larger diameter of the nerve, but the difference was not significant.

Figure 6

Measurement of threshold of action potential (A). The graph of action potential in the lab chart of PowerLab (B). The threshold of action potential of each group was compared to normal nerve. Pre-induced MSC coated cellulose/collagen nanofibrous conduit group showed significantly reduced than other groups. One way ANOVA between group, p < 0.0001. Values of **** p < 0.0001 were considered statistically significant (C).

Figure 6

Measurement of threshold of action potential (A). The graph of action potential in the lab chart of PowerLab (B). The threshold of action potential of each group was compared to normal nerve. Pre-induced MSC coated cellulose/collagen nanofibrous conduit group showed significantly reduced than other groups. One way ANOVA between group, p < 0.0001. Values of **** p < 0.0001 were considered statistically significant (C).

Figure 7

Pre-induced MSC showed better regeneration than other groups. (A) both groups I and II (treated with the MSC-coated nerve conduit) exhibited larger axons than the control group. (B) H&E and neurofilament immunostaining revealed smaller axonal diameters in the regenerated nerve fibers of the control group. (C) treatment with the MSC-coated nerve conduit alleviated this poor regeneration effect observed in the control group. Values of ** p < 0.01, *** p < 0.001, and **** p < 0.0001 were considered statistically significant.

Figure 7

Pre-induced MSC showed better regeneration than other groups. (A) both groups I and II (treated with the MSC-coated nerve conduit) exhibited larger axons than the control group. (B) H&E and neurofilament immunostaining revealed smaller axonal diameters in the regenerated nerve fibers of the control group. (C) treatment with the MSC-coated nerve conduit alleviated this poor regeneration effect observed in the control group. Values of ** p < 0.01, *** p < 0.001, and **** p < 0.0001 were considered statistically significant.

Figure 8

(A) The axon counts of three groups were similar (n = 255), but the gross size of an axon is higher in the pre-induced MSC group than other groups. (B) The thickness of myelin sheath of the pre-induced MSC group and MSC group show larger than the control media group. (C) reveals that thickness of myelin sheath of the pre-induced MSC is highest among three groups. ** p < 0.01 were considered statistically significant.

Figure 8

(A) The axon counts of three groups were similar (n = 255), but the gross size of an axon is higher in the pre-induced MSC group than other groups. (B) The thickness of myelin sheath of the pre-induced MSC group and MSC group show larger than the control media group. (C) reveals that thickness of myelin sheath of the pre-induced MSC is highest among three groups. ** p < 0.01 were considered statistically significant.

Figure 9

Fabrication schematic of cellulose/collagen nanofibrous conduit scaffold.

Figure 10

The schematic view of conduit.