title: Neuronal Networks on Nanocellulose Scaffolds.
abstract: Proliferation, integration, and neurite extension of PC12 cells, a widely used culture model for cholinergic neurons, were studied in nanocellulose scaffolds biosynthesized by Gluconacetobacter xylinus to allow a three-dimensional (3D) extension of neurites better mimicking neuronal networks in tissue. The interaction with control scaffolds was compared with cationized nanocellulose (trimethyl ammonium betahydroxy propyl [TMAHP] cellulose) to investigate the impact of surface charges on the cell interaction mechanisms. Furthermore, coatings with extracellular matrix proteins (collagen, fibronectin, and laminin) were investigated to determine the importance of integrin-mediated cell attachment. Cell proliferation was evaluated by a cellular proliferation assay, while cell integration and neurite propagation were studied by simultaneous label-free Coherent anti-Stokes Raman Scattering and second harmonic generation microscopy, providing 3D images of PC12 cells and arrangement of nanocellulose fibrils, respectively. Cell attachment and proliferation were enhanced by TMAHP modification, but not by protein coating. Protein coating instead promoted active interaction between the cells and the scaffold, hence lateral cell migration and integration. Irrespective of surface modification, deepest cell integration measured was one to two cell layers, whereas neurites have a capacity to integrate deeper than the cell bodies in the scaffold due to their fine dimensions and amoeba-like migration pattern. Neurites with lengths of >50 μm were observed, successfully connecting individual cells and cell clusters. In conclusion, TMAHP-modified nanocellulose scaffolds promote initial cellular scaffold adhesion, which combined with additional cell-scaffold treatments enables further formation of 3D neuronal networks.
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology , Göteborg, Sweden .
authors.forename: Malin
authors.initials: M
authors.lastname: Jonsson
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology , Göteborg, Sweden .
authors.forename: Christian
authors.initials: C
authors.lastname: Brackmann
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology , Göteborg, Sweden .
authors.forename: Maja
authors.initials: M
authors.lastname: Puchades
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology , Göteborg, Sweden .
authors.forename: Karoline
authors.initials: K
authors.lastname: Brattås
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology , Göteborg, Sweden .
authors.forename: Andrew
authors.initials: A
authors.lastname: Ewing
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology , Göteborg, Sweden .
authors.forename: Paul
authors.initials: P
authors.lastname: Gatenholm
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology , Göteborg, Sweden .
authors.forename: Annika
authors.initials: A
authors.lastname: Enejder
mesh_terms: D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002482:Cellulose / Q000494:pharmacology*; D003094:Collagen / Q000494:pharmacology; D008853:Microscopy; D053758:Nanoparticles / Q000737:chemistry*; D009415:Nerve Net / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D016716:PC12 Cells; D051381:Rats; D013059:Spectrum Analysis, Raman; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D002482:Cellulose; D003094:Collagen