3D Differentiation of Neural Stem Cells in Macroporous Photopolymerizable Hydrogel Scaffolds

<div><p>Neural stem/progenitor cells (NSPCs) are the stem cell of the adult central nervous system (CNS). These cells are able to differentiate into the major cell types found in the CNS (neurons, oligodendrocytes, astrocytes), thus NSPCs are the mechanism by which the adult CNS could potentially regenerate after injury or disorder. Microenviromental factors are critical for guiding NSPC differentiation and are thus important for neural tissue engineering. In this study, D-mannitol crystals were mixed with photocrosslinkable methacrylamide chitosan (MAC) as a porogen to enhance pore size during hydrogel formation. D-mannitol was admixed to MAC at 5, 10 and 20 wt% D-mannitol per total initial hydrogel weight. D-mannitol crystals were observed to dissolve and leave the scaffold within 1 hr. Quantification of resulting average pore sizes showed that D-mannitol addition resulted in larger average pore size (5 wt%, 4060±160 µm², 10 wt%, 6330±1160 µm², 20 wt%, 7600±1550 µm²) compared with controls (0 wt%, 3150±220 µm²). Oxygen diffusion studies demonstrated that larger average pore area resulted in enhanced oxygen diffusion through scaffolds. Finally, the differentiation responses of NSPCs to phenotypic differentiation conditions were studied for neurons, astrocytes and oligodendrocytes in hydrogels of varied porosity over 14 d. Quantification of total cell numbers at day 7 and 14, showed that cell numbers decreased with increased porosity and over the length of the culture. At day 14 immunohistochemistry quantification for primary cell types demonstrated significant differentiation to the desired cells types, and that total percentages of each cell type was greatest when scaffolds were more porous. These results suggest that larger pore sizes in MAC hydrogels effectively promote NSPC 3D differentiation.</p> </div

Pore size analysis of MAC scaffolds.

<p>(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 <i>post hoc</i> 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.</p

Figure 1

<p>(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.</p

Multiphoton confocal images of fluorescence staining for neurons, oligodendrocytes and astrocytes in 10 wt% scaffolds obtained at the center region of whole scaffolds.

<p>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.</p

Fluorescence staining results for NSPC differentiation.

<p>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.</p

Rheology and swelling results

<p>Letters denote significance by single factor ANOVA with Tukey’s <i>post hoc</i> analysis (p<0.01).</p

Oxygen diffusion data through 0, 5, 10, 20 wt% MAC scaffolds over 8 hrs.

<p>Mean ± SD with n = 3.</p

Oxygen diffusion in macroporous MAC scaffolds.

<p>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.</p

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.

<p>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.</p

Specific Immobilization of Biotinylated Fusion Proteins NGF and Sema3A Utilizing a Photo-Cross-Linkable Diazirine Compound for Controlling Neurite Extension

In this study we report the successful synthesis of <i>N</i>-(2-mercaptoethyl)-3-(3-methyl-3<i>H</i>-diazirine-3-yl) propanamide (N-MCEP-diazirine), with sulfhydryl and amine photoreactive ends to allow recombinant protein tethering to chitosan films. This regimen allows mimicry of the physiological endeavor of axon pathfinding in the nervous system where neurons rely on cues for guidance during development and regeneration. Our strategy incorporates strong covalent and noncovalent interactions, utilizing N-MCEP-diazirine, maleimide–streptavidin complex, and two custom biotinylated-fusion proteins, nerve growth factor (bNGF), and semaphorin3A (bSema3A). Synthetic yield of N-MCEP-diazirine was 87.3 ± 1.9%. Characteristic absorbance decrease at 348 nm after N-MCEP-diazirine exposure to UV validated the photochemical properties of the diazirine moiety, and the attachment of cross-linker to chitosan films was verified with Fourier transform infrared spectroscopy (FTIR). Fluorescence techniques showed no significant difference in the detection of immobilized proteins compared to absorbing the proteins to films (<i>p</i> < 0.05); however, <i>in vitro</i> outgrowth of dorsal root ganglia (DRG) was more responsive to immobilized bNGF and bSema3A compared to adsorbed bNGF and bSema3A over a 5 day period. Immobilized bNGF significantly increased DRG length over time (<i>p</i> < 0.0001), but adsorbed bNGF did not increase in axon extension from day 1 to day 5 (<i>p</i> = 0.4476). Immobilized bSema3A showed a significant decrease in neurite length (524.42 ± 57.31 μm) at day 5 compared to adsorbed bSema3A (969.13 ± 57.31 μm). These results demonstrate the superiority of our immobilization approach to protein adsorption because biotinylated-fusion proteins maintain their active confirmation and their tethering can be spatially controlled via a UV activated N-MCEP-diazirine cross-linker