Figure 1.
Schematic illustration of the basic steps in fabricating the NGF–CMSs/CCH.
Figure 2.
Microstructure appearance of the NGF–CMSs, CCH, and NGF–CMSs/CCH.
Scanning electron microscopy images of the NGF–CMSs (A and B) show relative rough surface and spherical shape without hollows or deformations. Scanning electron microscopy images of the CCH scaffold in a transverse section (C) show that the microchannels were arranged in a honeycomb-like pattern. The longitudinal section of the CCH (D) shows the longitudinally oriented microchannels. Scanning electron microscopy images of the NGF–CMSs/CCH in a transverse section (E) show slightly different morphologies with pores, which were less regular and smaller than that of the CCH scaffold. The longitudinal section of the NGF–CMSs/CCH shows that the NGF–CMSs were evenly distributed throughout the longitudinally oriented microchannels which did not influence the microstructures of the scaffolds.
Figure 3.
In vitro release kinetics of NGF from the NGF/CCH and NGF–CMSs/CCH over 28 days.
All data represent mean ± SD (n = 3). *p<0.05 between NGF/CCH and NGF–CMSs/CCH.
Figure 4.
All data represent mean ± SD. *p<0.05 between control and others (NGF/CCH and NGF–CMSs/CCH). #p<0.05 between NGF/CCH and NGF–CMSs/CCH.
Figure 5.
The PC12 cells which were co-cultured for 1, 7, 14, and 28 days in the CCH group (without NGF), NGF/CCH group (NGF amount: 100 ng), and NGF–CMSs/CCH group (microspheres amount: 70 mg, which contain 100 ng NGF). All data represent mean ± SD. *p<0.05 between CCH and others (NGF/CCH and NGF–CMSs/CCH). #p<0.05 between NGF/CCH and NGF–CMSs/CCH.
Figure 6.
Electrophysiological assessments.
All data represent mean ± SD. *p<0.05 between autograft and others (CCH, NGF/CCH, and NGF–CMSs/CCH). #p<0.05 between NGF–CMSs/CCH and others (CCH and NGF/CCH). &p<0.05 between NGF/CCH and CCH.
Figure 7.
Fluoro-Gold (FG) retrograde tracing.
Micrographs of FG-labeled motoneurons in spinal cord (transverse sections: A1, B1, C1, and D1; longitudinal sections: A2, B2, C2, and D2) and sensory neurons in dorsal root ganglions (transverse sections: A3, B3, C3, and D3) in the autograft group (A1–A3), NGF–CMSs/CCH group (B1–B3), NGF/CCH group (C1–C3), and CCH group (D1–D3) at 16 weeks after nerve-bridging operation. The total number of FG-labeled motoneurons and sensory neurons in each group were shown in (E) and (F). All data represent mean ± SD. *p<0.05 between autograft and others (CCH, NGF/CCH, and NGF–CMSs/CCH). #p<0.05 between NGF–CMSs/CCH and others (CCH and NGF/CCH). &p<0.05 between NGF/CCH and CCH. Scale bar = 100 µm.
Figure 8.
Morphometric analyses of regenerated nerves.
Toluidine blue staining of regenerated axons at the distal portion (A–D) and transmission electron micrographs of regenerated axons at the distal portion (E–H) in the autograft group (A and E), NGF–CMSs/CCH composite group (B and F), NGF/CCH group (C and G), and CCH group (D and H). Morphometric evaluations of regenerated nerves at the distal portion were performed at 16 weeks after nerve-bridging operation. (I) The number of myelinated axons (Mtot), (J) the total area of regenerated axons (Atot), (K) the diameter of myelinated axons, and (L) the G-ratio were measured from the distal section of the regenerated nerves. All data represent mean ± SD. *p<0.05 between autograft and others (CCH, NGF/CCH, and NGF–CMSs/CCH). #p<0.05 between NGF–CMSs/CCH and others (CCH and NGF/CCH). &p<0.05 between NGF/CCH and CCH. Scale bar = 10 µm for A, B, C, and D; scale bar = 1µm for E, F, G, and H.