Nano-Patterned Poly-ε-caprolactone with Controlled Release of Retinoic Acid and Nerve Growth Factor for Neuronal Regeneration

Abstract

A combination of nanotopography and controlled release could be a potential treatment for damages to the central and peripheral nervous system. Synergistic effects of both physical and chemical guidance were more effective than individual cues in the directional promotion of neurite outgrowth [1]. Hypothesizing that synergistic effect will enhance neuronal differentiation of human mesenchymal stem cells (hMSCs), a fabrication method for neurotrophic factors encapsulation in a nanopatterned (350nm-gratings) poly-ε-caprolactone (PCL) film was developed. Nanotopography directs hMSCs into neuronal lineage while controlled release of neurotrophic factors presents biochemical signals with temporal and spatial control. Preliminary results demonstrated synergistic effect on hMSC cultured on substrates with both nanotopographical and biochemical cues. The protein/drug encapsulated PCL films were fabricated by modified solvent casting methods in which the biologics were 'sandwiched' between two layers of PCL on a nanopatterned polydimethylsiloxane mold. Bovine serum albumin (BSA), RA and NGF were encapsulated into the films using this sandwich approach. BSA was first encapsulated to optimize the sustained release. Among different fabrication methods in BSA optimization, encapsulating solid BSA between 2 PCL layers resulted in most sustainable release. Surface topography remained intact after 10 days incubated in PBS with controlled release, verified through SEM. Continuous release of bioactive RA and NGF was observed over the 1 week release period. Human MSCs aligned and elongated along the nano-patterned films. Enhanced upregulation of neuronal gene such as microtubule associated protein (MAP2) and neurofilament (NFL) was observed in hMSCs cultured on combination cues, as compared to individual cues. Using quantitative PCR analysis, upregulation of MAP2 in hMSCs on the nanogratings with controlled release NGF was verified, highlighting the synergistic effect of both biochemical and nanotopographical cues in directing hMSC differentiation. This fabrication methodology will allow the delivery of multiple therapeutic cues for potential applications in neural regeneration.