Figure 1.
(A) Methodology for creating 3D porous MAC scaffolds and procedure for NSPC culture and differentiation in 3D environments. (B) Images of a 20% D-mannitol scaffold captured immediately after crosslinking and after PBS dissolution for 1 hr at 37°C.
Figure 2.
Oxygen diffusion in macroporous MAC scaffolds.
Oxygen diffusion device allowing us to measure depletion of oxygen in the top chamber as diffusion occurs through the gel into the oxygen free bottom chamber.
Figure 3.
Pore size analysis of MAC scaffolds.
(A) Microscope images of acellular MAC scaffolds with varying mass percentages of D-mannitol. (B) Pore sizes of MAC scaffolds with varied D-mannitol percentages. Letters denote significance by single factor ANOVA with Tukey’s post hoc analysis (p<0.001). (C) SEM images of MAC scaffolds with varying mass percentages of D-mannitol. Freeze-dried scaffolds collapse during the process, so the pore sizes are not directly comparable to those shown in A and B. Mean ± SD with n = 3.
Figure 4.
Oxygen diffusion data through 0, 5, 10, 20 wt% MAC scaffolds over 8 hrs.
Mean ± SD with n = 3.
Table 1.
Rheology and swelling results
Figure 5.
Fluorescence staining results for NSPC differentiation.
Quantification of IHC at day 14 shows that more porous scaffolds (up to 20 wt% D-mannitol initially) in (A) neuron specific media (IFN-γ) favor neurons. (B) Oligodendrocyte specific media (PDGF-AA) favor oligodendrocytes and (C) in astrocyte specific media (BMP-2) favor astrocytes. (D) Control media with no growth factors (-GF) as well as with proliferation growth factors (+EGF+FGF) maintain nestin expression (note: error bars are included but too small to see). Letters denote significance by single factor ANOVA (p<0.001). Mean ± SD with n = 3.
Figure 6.
Multiphoton confocal images of fluorescence staining for neurons, oligodendrocytes and astrocytes in 10 wt% scaffolds obtained at the center region of whole scaffolds.
Corresponding zoomed regions (white rectangle) for each image are provided below. Nuclei appear blue by Hoechst 33342, cell staining for each differentiation marker appear red by Alexa-Fluor 546.
Figure 7.
Total cell number at day 7 and 14 for porous scaffolds cultured in control (+EGF+FGF, -GF) and differentiation (INF-γ, PDGF-AA, BMP-2) media.
NSPCs were initially seeded at 200 × 103 cells/scaffold. *** denotes significance by two-factor ANOVA (p<0.0001). Mean ± SD with n = 3. All scaffolds were cultured for 1 d in expansion media (+EGF+FGF) then switched to conditions labeled in the caption.