Asymmetrical 3D Nanoceria Channel for Severe Neurological Defect Regeneration

Abstract

Inflammation and oxidative stress are major problems in peripheral nerve injury. Nanoceria can manipulate antioxidant factor expression, stimulate angiogenesis, and assist in axonal regeneration. We fabricate collagen/nanoceria/polycaprolactone (COL/NC/PCL) conduit by asymmetrical three-dimensional manufacture and find that this scaffold successfully improves Schwann cell proliferation, adhesion, and neural expression. In a 15-mm rat sciatic nerve defect model, we further confirm that the COL/NC/PCL conduit markedly alleviates inflammation and oxidative stress, improves microvessel growth, and contributes to functional, electrophysiological, and morphological nerve restoration in the long term. Our findings provide compelling evidence for future research in antioxidant nerve conduit for severe neurological defects.

Keywords: Biomaterials; Nanotechnology; Neurosurgery.

Graphical abstract

Figure 1

Schematic Illustration of COL/NC/PCL Nerve Conduit Fabrication and Implantation into a Rat Model (A) Asymmetrical three-dimensional layer-by-layer manufacture of COL/NC/PCL nerve conduit. It was composed of three layers: the innermost NC/PCL mixed layer, the outermost COL layer, and the middle PCL layer. A tube mold was rolling counterclockwise, under a sprayer that injected different solutions layer by layer on the rolling tube. A microneedle on the tube assured even pore size that allowed free exchanges of nutrients into the conduit. The schematic illustration showed Schwann cell adhesion to the innermost layer and fibroblast detachment from the outermost layer. (B) Rough innermost layer; scale bar, 10 μm. (C) Smooth outermost layer; scale bar, 10 μm. (D) Multilayered structure and an increasing gradient change in roughness inside out; scale bar, 20 μm. (E) Microporous structure in the COL/NC/PCL nerve conduit; scale bar, 5 μm.

Figure 2

Schwann Cell Morphology in Different Conduits (A–U) Cell morphology in different nerve conduits evaluated by SEM (A–I) and immunofluorescence (J–U). (A–C) Cell morphology in COL/NC/PCL conduit. (D–F) Cell morphology in NC/PCL conduit. (G–I) Cell morphology in PCL conduit. Scale bars, 100 μm in (A, D, and G); 50 μm in (B, E, and H); and 20 μm in (C, F, and I). (J–M) Phalloidin staining showing cell attachment in COL/NC/PCL conduit. (J, N, and R) Phalloidin staining; scale bars, 25 μm. (K, O, and S) DAPI staining; scale bars, 25 μm. (L, P, and T) Merged images; scale bars, 25 μm. (M, Q, and U) 3D display for cell attachment in different conduits; scale bars, 50 μm. (V) Cell protuberances in different conduits, *p < 0.05 compared with COL/NC/PCL, ^#p < 0.05 compared with NC/PCL.

Figure 3

Immunofluorescence of Ki67 and GFAP (A–D) Ki67 expression in COL/NC/PCL. (E–H) Ki67 expression in NC/PCL. (I–L) Ki67 expression in PCL. (M–P) GFAP expression in COL/NC/PCL. (Q–T) GFAP expression in NC/PCL. (U–X) GFAP expression in PCL. (A, E, and I) Ki67 staining; scale bars, 25 μm. (M, Q, and U) GFAP staining; scale bars, 25 μm. (B, F, J, N, R, and V) DAPI staining; scale bars, 25 μm. (C, G, K, O, S, and W) Merged images; scale bars, 25 μm. (D, H, L, P, T, and X) 3D display; scale bars, 50 μm. GFAP, glial fibrillary acidic protein.

Figure 4

Immunofluorescence of S100 and Tuj1 (A–D) S100 expression in COL/NC/PCL. (E–H) S100 expression in NC/PCL. (I–L) S100 expression in PCL. (M–P) Tuj1 expression in COL/NC/PCL. (Q–T) Tuj1 expression in NC/PCL. (U–X) Tuj1 expression in PCL. (A, E, and I) S100 staining; scale bars, 25 μm. (M, Q, and U) Tuj1 staining; scale bars, 25 μm. (B, F, J, N, R, and V) DAPI staining; scale bars, 25 μm. (C, G, K, O, S, and W) Merged images; scale bars, 25 μm. (D, H, L, P, T, and X) 3D display, scale bars, 50 μm.

Figure 5

Immunofluorescence of MBP (A–D) MBP expression in COL/NC/PCL. (E–H) MBP expression in NC/PCL. (I–L) MBP expression in PCL. (A, E, and I) MBP staining; scale bars, 25 μm. (B, F, and J) DAPI staining; scale bars, 25 μm. (C, G, and K) Merged images; scale bars, 25 μm. (D, H, and L) 3D display; scale bars, 50 μm. (M) Western blots of Ki67, S100, Tuj1, and MBP compared among COL/NC/PCL, NC/PCL, and PCL conduits. (N) Relative Ki67 mRNA level. (O) Relative S100 mRNA level. (P) Relative Tuj1 mRNA level. (Q) Relative MBP mRNA level. *p < 0.05 compared with COL/NC/PCL, ^#p < 0.05 compared with NC/PCL.

Figure 6

TEM for Axonal Regeneration and Remyelination State at 18 Weeks Postoperatively (A–C) COL/NC/PCL conduit. (D–F) NC/PCL conduit. (G–I) PCL conduit. (J–L) Autograft. Scale bars, 10 μm in (A, D, G, and J); 2 μm in (B, E, H, and K); and 1 μm in (C, F, I, and L).

Figure 7

Triple Immunofluorescence of Regenerated Nerve Tissues Showing Axonal Restoration at 18 Weeks Postoperatively (A–D) COL/NC/PCL. (E–H) NC/PCL. (I–L) PCL. (M–P) Autograft. (A, E, I, and M) DAPI staining. (B, F, J, and N) NF200 staining. (C, G, K, and O) Tuj1 staining. (D, H, L, and P) Merged images. Scale bars, 100 μm.

Figure 8

Triple Immunofluorescence Showing Schwann Cell Viability and Remyelination at 18 Weeks Postoperatively (A–X) (A–C and M–O) COL/NC/PCL. (D–F and P–R) NC/PCL. (G–I and S–U) PCL. (J–L and V–X) Autograft. (A, D, G, J, M, P, S, and V) DAPI staining. (B, E, H, and K) MBP staining. (N, Q, T, and W) S100 staining. (C, F, I, L, O, R, U, and X) Merged images. Scale bars, 100 μm.