Figure 1.
Fabrication and characterization of biofunctionalized and aligned silk nanofibers.
A) Fibroin solution was mixed (or not) with NGF and CNTF and electrospun as described in material and methods. B) SEM image of aligned silk nanofibers on glass slides; Alignment and mean diameter of electrospun fibers, measured as described in material and methods; Scale bar: 5 µm. C) Oblique plot and surface profile of fibroin nanofibers electrospun on a glass slide.
Figure 2.
Growth factors release from electrospun functionalized silk and stability.
A) No NGF was detected from the silk electrospun after 5 days in PBS. B) As NGF, no CNTF was detected from the electrospun. Degradation of growth factors was observed in PBS with 0.1% BSA. After 5 days, more than 50% of NGF could be detected unlike 10% for CNTF.
Figure 3.
PC12 differentiation induced by NGF-functionalized nanofibers.
PC12 cells grown for 8 days on: glass slide a), fibroin nanofibers b), fibroin nanofibers with NGF (50 ng. mL-1) in culture medium c) and NGF-functionalized fibroin nanofibers d); scale bar: 25µm. Number of neurites per cell e) and neurites length f). White bars: NGF in solution, grey bars: NGF-functionalized fibroin and black bars: fibroin. Quantification was performed as described in material and methods. Standard deviations are shown as error bars. Significant differences (ns: no significance, * p<0,05; *** p<0,001)
Figure 4.
DRG neurons adhesion and growth on electrospun fibroin nanofibers.
SEM observation of DRG cells adhering on nanofibers (A) and establishing tight contact (B and C). BIII tubulin staining of neurons growing on random (D) and aligned (E) nanofibers. BIII tubulin (red) and actin (green) of neuron growth cones on random (F) and aligned (G) nanofibers. Image acquisition performed after 5 days of culture. Scale bars: A 10µm, B 5µm, C 2µm, D, E, F and G 10µm. Images A, B and C have been recolored for sake of clarity.
Figure 5.
Bioactivity of functionalized electrospun fibroin nanofibers on DRG neurons growth.
DRGs cells were seeded on either unfunctionalized (A and e), NGF-functionalized (B and f), CNTF (C and g) or NGF and CNTF-functionalized nanofibers (D and h) and neurons were stained with anti BIII tubulin antibody. Fluorescence images were acquired after 5 days of culture (A, B, C and D). Higher magnification of individual growing neurons (e, f, g and h) were acquired 3 days after seeding, to prevent overlapping of neurons currently observed after a longer time in culture. For details see material and methods. Scale bars: A, B, C, D: 100µm; e, f, g, h: 20 µm.
Table 1.
Quantification of nanofibers fibers bioactivity on dissociated DRGs cell culture.
Figure 6.
Bioactivity of multifunctionalized nanofibers in organotypic DRG culture.
Fœtal DRG were seeded on unfunctionalized (A, B) or multifunctionalized (C–H) nanofibers. Images were acquired after 12 days (A, C) and 4 weeks (B, D–H) of culture. Fluorescence (B, D, E, F) and scanning laser confocal microscopy (G, H) images were obtained as described in material and methods. DRG neural tubulin was stained with anti-BIII tubulin (red), glial cells actin was stained with phalloidin (green) and nuclei with DAPI (blue). Scale bars: A, B, C, D: 250 µm; E: 10µm and G: 50µm.
Figure 7.
Development of a multifunctionalized nanofiber-based tube for in vivo implantation.
Fibroin scaffold is collected at the end of the electrospinning process and rolled up on teflon sticks (A), higher magnification of the designed fibroin tube (B), SEM observation of a cross section of the fibroin tube (C), higher magnification highlighting the alignment of nanofibers inside the tube (D) and nanofiber-based tube sutured to a rat sciatic nerve (E). Scale bar: 1 cm A), 1 mm B and E), 200µm C) and 30µm D). For details see material and methods.