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.