(558e) Neural Stem Cell 3D Neuronal Differentiation in Fluorinated Methacrylamide Chitosan Hydrogels

Oxygen
plays a vital role in the metabolism of living organisms and in tissue
regrowth. Tissue engineering offers a potential solution to regenerate many
tissues in the body including the central nervous system (CNS). Within the CNS,
neural stem cells (NSCs) have been isolated that offer potential cellular
treatments for neurodegenerative diseases, stroke, traumatic brain injury, and
spinal cord injury. We recently invented a biocompatible photocrosslinkable
chitosan hydrogel modified
with perfluorocarbons (PFCs), which we call perfluorocarbon methacrylamide chitosan (MACF) that is able to uptake and release
beneficial levels of oxygen to cells and tissues. The overall objective of this
work is to demonstrate how MACFs can be utilized in 3D tissue engineered
scaffolds to enhance cellularity and NSC differentiation.

In
this study a series of novel, biocompatible, oxygen rich hydrogel scaffolds
have been developed and synthesized by incorporating several PFCs to photopolymerizable methacrylamide
chitosan hydrogels via
Schiff base, nucleophilic substitution followed by
radical polymerization to form hydrogels. The synthesized fluorinated methacrylamide chitosan (MACF) hydrogels were characterized by high resolution ¹⁹F
NMR and demonstrated the desired degree of fluorine substitution (35-45%). MACF
oxygen adsorption and desorption studies demonstrated that MACF can be easily
regenerated by reloading with oxygen once release is complete for at least
three cycles with minimal change in properties. To study how oxygen rich
hydrogels affect NSC survival and differentiation, NSCs were seeded and
cultured in oxygenated and unoxygenated MACF hydrogels (MAC(Ali5)F with 5
aliphatic fluorine substitutions, MAC(Ar5F) with 5 aromatic fluorine
substitutions and MAC(Ali15) with 15 aliphatic fluorine substitutions) and
compared to nonfluorinated MAC hydrogels.
After 8 days of differentiation and culture (in neuron specific medium), total
cell number and neuronal differentiation was quantified. Results have revealed
that MACFs support more cells than unfluorinated
controls, especially in the interior of the scaffolds. Significant neuronal
differentiation has been observed in all scaffolds, and we are currently
studying neuronal extension and process formation.