Neurons sense nanoscale roughness with nanometer sensitivity

Abstract

The interaction between cells and nanostructured materials is attracting increasing interest, because of the possibility to open up novel concepts for the design of smart nanobiomaterials with active biological functionalities. In this frame we investigated the response of human neuroblastoma cell line (SH-SY5Y) to gold surfaces with different levels of nanoroughness. To achieve a precise control of the nanoroughness with nanometer resolution, we exploited a wet chemistry approach based on spontaneous galvanic displacement reaction. We demonstrated that neurons sense and actively respond to the surface nanotopography, with a surprising sensitivity to variations of few nanometers. We showed that focal adhesion complexes, which allow cellular sensing, are strongly affected by nanostructured surfaces, leading to a marked decrease in cell adhesion. Moreover, cells adherent on nanorough surfaces exhibit loss of neuron polarity, Golgi apparatus fragmentation, nuclear condensation, and actin cytoskeleton that is not functionally organized. Apoptosis/necrosis assays established that nanoscale features induce cell death by necrosis, with a trend directly related to roughness values. Finally, by seeding SH-SY5Y cells onto micropatterned flat and nanorough gold surfaces, we demonstrated the possibility to realize substrates with cytophilic or cytophobic behavior, simply by fine-tuning their surface topography at nanometer scale. Specific and functional adhesion of cells occurred only onto flat gold stripes, with a clear self-alignment of neurons, delivering a simple and elegant approach for the design and development of biomaterials with precise nanostructuretriggered biological responses.nanobiointeractions | nanostructures | patterning T he potential of nanomaterials to trigger specific cellular responses, such as interference and/or activation of defined pathways (1-3), is promising for the development of many important scientific fields, such as regenerative medicine, biotechnology, drug delivery, and nanotoxicity assessment. Initially, cellsmaterials interactions were tackled only from a chemical point of view, because environmental sensing by cells involves specific binding between cellular receptors and ECM ligands. However, recently there has been increasing evidence that the biological response is also affected by the physical properties of the material (4). In particular, it has been demonstrated that cells are influenced by the substrate topography (5, 6), rigidity (7, 8), anisotropy (9, 10), surface charge (11, 12), and wettability (13,14). From this perspective, cellular response to external stimuli goes far beyond the bare ability of the cell to chemically sense specific ECM ligands and includes a wide range of physical cues that are generated at, or act on, the interface between cells and the surrounding environment. For instance, it has been observed that micrometer-scale roughness may affect cell proliferation and morphology (15,16), because it provides a quasi-biomimetic microenvironment to the cells. How...

Substrate topography: A valuable in vitro tool, but a clinical red herring for in vivo tenogenesis

“…electrospun polymeric fibres, extruded collagen fibres and isoelectrically focused collagen fibres) have been shown to maintain tenocyte phenotype and to differentiate stem cells towards tenogenic lineage in vitro and to induce acceptable regeneration in preclinical models, none of these technologies offers precise control over the spatial distribution of the fibres. Imprinting technologies, on the other hand, have demonstrated a diverse effect on a range of permanently differentiated and stem cell functions, including adhesion, orientation, secretome expression and lineage commitment [41][42][43][44][45][46][47][48] and offer significantly greater control over feature dimension and spacing. Specifically to tendon repair, such technologies have been shown to maintain tenocyte phenotype [38]; to promote aligned tendon-specific ECM deposition [39]; and to differentiate stem cells towards tenogenic lineage [40].…”

Stochastic nanoroughness modulates neuron–astrocyte interactions and function via mechanosensing cation channels

“…However, in addition to a biological function, cells and macromolecules provide a physically defined environment (19,20), and we postulate a significant role for topography in neural development. Studies to date have focused on the effects of microscale topography, deterministic roughness, and substrate chemistry on neurite outgrowth and neuronal function (7,(21)(22)(23). However, the influence of ECM-like nanotopography on neuronal development and fate is a realm that has not been investigated thus far.…”

“…However, developmental processes such as axon pathfinding, synapse formation, nervous system patterning, neuronal plasticity, and degeneration fail to be explained solely on the basis of soluble factors. There is increasing evidence that physical variables such as the stiffness of a cellular environment influence cell development (7)(8)(9)(10)(11)(12). However, the cells of the brain tissue reside in a soft environment that is rich in polysaccharides (13,14).…”

Estudo das células Neuro2A sobre os biomateriais PCL e PLLA

“…Além disso, alguns experimentos in vitro demonstraram que a utilização de fatores de crescimento e outras substâncias como o interferon e acetato de glatiramer provocam a proliferação celular [7,8] . Por outro lado, as características intrínsecas dos biomateriais, como a especificidade química da superfície, elétrica, hidrofobicidade e topografia, podem influenciar na adesão e crescimento celular [1,3,4,9,10] . Ainda, observa-se que dentre os biomateriais utilizados para o cultivo celular, os poli α-hidróxi ácidos, vêm se tornando uma das famílias de polímeros mais promissoras e estudadas na área dos materiais bioreabsorvíveis, destacando-se o poli ácido glicólico (PGA) e o poli caprolactona (PCL), por atuarem positivamente em vários ensaios in vivo e in vitro [2,9,[11][12][13][14][15][16] .…”

“…Sabe-se que a adesão, migração, diferenciação e a proliferação celular sobre determinado substrato depende em parte das propriedades intrínsecas de cada tipo celular, bem como, o tipo de material utilizado [1][2][3][4] . Dentre os produtos celulares, pode-se ressaltar a síntese de moléculas de adesão celular, como laminina, fibronectina e colágeno, importantes para o cultivo e o crescimento celular [5,6] .…”