Anodic Aluminum Oxide (AAO) Membranes for Neurite Outgrowth
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Anodic Aluminum Oxide (AAO) Membranes for Neurite Outgrowth Meghan E. Casey1, Anthony P. Ventura2,3, Wojciech Z. Misiolek2,3 and Sabrina Jedlicka1,2,4 1
Bioengineering, 2Materials Science and Engineering, 3Institute for Metal Forming, 4Center for Advanced Materials and Nanotechnology Lehigh University, Bethlehem, PA 18015 USA
ABSTRACT Anodic aluminum oxide (AAO) membranes were fabricated in a mild two-step anodization procedure. The voltage was varied during both anodization steps to control the pore size and morphology of the AAO membranes. Pore sizes ranged from 34 nm to 117 nm. Characterization of the pore structure was performed by scanning electron microscopy (SEM). To assess the potential of the AAO membranes as a neuronal differentiation platform, C17.2 neural stem cells (NSCs), an immortalized and multipotent cell line, were used. The NSCs were forced to differentiate via serum-withdrawal. Cellular growth was characterized by immunocytochemistry (ICC) and SEM. ImageJ software was used to obtain phenotypic cell counts and neurite outgrowth lengths. Results indicate a highly tunable correlation between AAO nanopore sizes and differentiated cell populations. By selecting AAO membranes with specific pore size ranges, control of neuronal network density and neurite outgrowth length was achieved. INTRODUCTION The ability to generate and control neuronal growth in vitro from stem cell precursors is a growing area of interest in biomedicine. Neuronal development occurs in various stages from immature precursor cells to fully integrated and functionally mature neurons [1]. These developmental steps are classified into two categories: activity independent and activity dependent. Independent landmarks are thought to be genetically determined and include neuronal differentiation, migration and axon guidance [2]. Activity dependent stages of neuronal growth are heavily regulated by secreted molecules such as hormones and neurotransmitters [2]. The overall effects of the secreted molecules in vivo are well researched; however, the effects on in vitro differentiation are not fully understood. Researchers are unable to identify and measure the small molecules in vitro, as the secreted hormones are absorbed by neighboring cells. Therefore, a substantial opportunity exists in neuronal interface research to develop a material platform that allows for both the proliferation and differentiation of stem cells into neurons and the ability to quantify the secretome of neuronal cells. C17.2 neural stem cells (NSCs) are an immortalized and multipotent cell line established by Snyder et al. [3,4]. Derived from the external germinal layer of neonatal mouse cerebellum, C17.2 neural precursors have been show to successfully implant into mouse germinal zones [4]. The NSCs integrate into the implanted tissue and contribute to cerebellum development [4].
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Because C17.2 cells are functional in vivo, they are a relevant cell model for studying neuronal platforms. AAO membranes are biocompatible and composed of highly-ordered nanopores
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