INTRODUCTIONWe recently set up nanocomposite, self standing scaffolds recapitulating nanotopographical features of the neural environment by combining the biocompatibility of a poly-L-lactic (PLLA) matrix with the electrical,... [ view full abstract ]
INTRODUCTION
We recently set up nanocomposite, self standing scaffolds recapitulating nanotopographical features of the neural environment by combining the biocompatibility of a poly-L-lactic (PLLA) matrix with the electrical, mechanical and chemical properties of carbon nanostructures. In the prototype system, dispersed carbon nanotubes (CNTs) in PLLA proved to support differentiation of human SH-SY5Y neuronal precursors. Then, we used human circulating multipotent cells (hCMCs), which are free from ethical restrictions, not genetically transformed and easily accessible, as a source for autologous and safe transplant. In order to further optimize scaffold features, we tested varying nanostructure concentrations and types comparing as nanofillers carbon nanohorns (CNHs) and reduced graphene oxide (rGO) to CNT-PLLA scaffolds.
MATERIAL AND METHODS
Nanocomposite scaffolds were prepared using CNTs or CNHs or rGO dispersed at 0.25% or 5% in the PLLA matrix. Biocompatibility and cell proliferation were assayed by LDH release and resazurin methods. hCMCs were isolated from volunteer healthy donors by Ficoll density gradient separation and characterized by flow cytometry (FCM) and qPCR analysis. Neuronal commitment was followed up by both fluorescence microscopy and evaluation of the expression of neural marker genes (Nestin, βIII tubulin/TUBβ3, Microtubule-associated protein 2/MAP2 and L1 CAM).
RESULTS
In presence of proper growth factors and media, hCMCs can be alternatively committed toward adipocyte, osteoblast, myocyte or neuronal lineages. Intriguingly however, even in the absence of neurotrophins, hCMCs are committed towards neuronal lineage when cultured onto CNT-PLLA scaffold (see figure), as shown by both microscopy and qPCR evidences. Comparison among CNTs, CNHs and rGO at varying concentrations is on-going and preliminary evidence with SH-SY5Y is encouraging as all tested conditions proved to be fully biocompatible.
DISCUSSION
This work suggests that hCMCs grown and differentiated onto our biomimetic and conductive scaffolds may represent a self standing prototype for reparation and repopulation of neural tissue injuries as well as, in general, a safe and autologous source for regenerative medicine and tissue engineering applications. Further variation of the scaffold composition will hopefully help us to boost neuronal differentiation and to shed light on contributions of peculiar scaffold features (shape, stiffness, conductivity) to this path.
