Chitin Nerve Conduits with Three-Dimensional Spheroids of Mesenchymal Stem Cells from SD Rats Promote Peripheral Nerve Regeneration

Abstract

Peripheral nerve injury (PNI) is an unresolved medical problem with limited therapeutic effects. Epineurium neurorrhaphy is an important method for treating PNI in clinical application, but it is accompanied by inevitable complications such as the misconnection of nerve fibers and neuroma formation. Conduits small gap tubulization has been proved to be an effective suture method to replace the epineurium neurorrhaphy. In this study, we demonstrated a method for constructing peripheral nerve conduits based on the principle of chitosan acetylation. In addition, the micromorphology, mechanical properties and biocompatibility of the chitin nerve conduits formed by chitosan acetylation were further tested. The results showed chitin was a high-quality biological material for constructing nerve conduits. Previous reports have demonstrated that mesenchymal stem cells culture as spheroids can improve the therapeutic potential. In the present study, we used a hanging drop protocol to prepare bone marrow mesenchymal stem cell (BMSCs) spheroids. Meanwhile, spherical stem cells could express higher stemness-related genes. In the PNI rat model with small gap tubulization, BMSCs spheres exhibited a higher ability to improve sciatic nerve regeneration than BMSCs suspension. Chitin nerve conduits with BMSCs spheroids provide a promising therapy option for peripheral nerve regeneration.

Keywords: bone marrow mesenchymal stem cells; chitin; peripheral nerve conduits; peripheral nerve injury; spheroid.

Figure 1

The construction process of chitin nerve conduits.

Figure 2

Schematic diagram of each group of nerve conduits and implants.

Figure 3

Identification of BMSCs. (a) Typical vortex distribution of BMSCs. (b) Alizarin red S staining revealed that osteogenic capacity of BMSCs. (c) Oil Red O staining showed adipogenic differentiation of BMSCs. (d) Expression of BMSCs surface markers CD29, CD34, CD45, and CD90 detected by flow cytometry. Red lines indicated the blank control BMSCs, and green lines indicated the antibodies-bound BMSCs.

Figure 4

The morphology and stemness-related properties of BMSCs spheroids. (a) Schemes of hanging drop method to culture BMSCs spheroids. (b) Gross view of the fabrication process of BMSCs spheroids. (c) Representative image of BMSCs spheroids. Scale bar = 50 μm. (d) Immunofluorescence image of BMSCs using phalloidin and DAPI staining. Scale bar = 50 μm. (e–g) mRNA expression levels of Nanog, Sox2, and POU5F1 in different cultures of BMSCs. ** p < 0.01, vs. BMSCs group (Student’s t-test).

Figure 5

(a) Gross view of the chitin nerve conduit. (b) The bending image of nerve conduit. (c) The scanning electron microscopy image of surface morphology of chitin conduit.

Figure 6

Tensile test of the chitin nerve conduit. (a–c) The conduit was performing tensile test. (d) Stress–strain curve of the chitin nerve conduit.

Figure 7

FTIR spectra of chitin and chitosan films.

Figure 8

Photograph of the water drop in contact with the surface of chitin film.

Figure 9

Live/dead staining images of SCs seeded on chitin film. (a) The live SCs were labeled with Calcein-AM (green). (b) The dead SCs were labeled with PI (red). (c) Merged image of live/dead staining. Scale bar = 50 μm.

Figure 10

Motor function 4 weeks and 8 weeks after surgery. (a) Representative footprints images of right (injured) hind and left (normal) hind paw at 8 weeks after operation. (b) The sciatic function index (SFI) recorded at 4 weeks and 8 weeks after operation. Data are expressed as median with interquartile range (n = 5 for each group). * p < 0.05, ** p < 0.01, vs. Control group; # p < 0.05, vs. BMSCs group (non-parametric test followed by Kolmogorov–Smirnov and Shapiro–Wilk tests).

Figure 11

Electrophysiological examinations conducted 8 weeks after surgery. (a) Representative CMAP waveform at the operation side in each group. (b) Statistical analysis of CMAP latency. (c) Statistical analysis of CMAP amplitude. Data are expressed as mean ± SEM (n = 5 for each group). ** p < 0.01, vs. Control group; # p < 0.05, vs. BMSCs group (One-way analysis of variance followed by Kolmogorov–Smirnov and Shapiro–Wilk tests).

Figure 12

Histological evaluation of the regenerated nerve fibers. (a) TEM images of the regenerated sciatic nerve transverse sections 8 weeks postoperatively. (b) Diameters of the regenerated nerve fibers. Data are expressed as median with interquartile range (n = 5 for each group). (c) Thickness of remyelination sheath. Data are expressed as mean ± SEM (n = 5 for each group). * p < 0.05, vs. Control group; # p < 0.05, vs. BMSCs group. Diameters of the regenerated nerve fibers (non-parametric test followed by Kolmogorov–Smirnov and Shapiro–Wilk tests). Thickness of remyelination sheath. One-way analysis of variance followed by Kolmogorov–Smirnov and Shapiro–Wilk tests).