Biomechanics of Macrophages on Disordered Surface Nanotopography.

Macrophages are involved in every stage of the innate/inflammatory immune responses in the body tissues, including the resolution of the reaction, and they do so in close collaboration with the extracellular matrix (ECM). Simplified substrates with nanotopographical features attempt to mimic the structural properties of the ECM to clarify the functional features of the interaction of the ECM with macrophages. We still have a limited understanding of the macrophage behavior upon interaction with disordered nanotopography, especially with features smaller than 10 nm. Here, we combine atomic force microscopy (AFM), finite element modeling (FEM), and quantitative biochemical approaches in order to understand the mechanotransduction from the nanostructured surface into cellular responses. AFM experiments show a decrease of macrophage stiffness, measured with the Young's modulus, as a biomechanical response to a nanostructured (ns-) ZrOx surface. FEM experiments suggest that ZrOx surfaces with increasing roughness represent weaker mechanical boundary conditions. The mechanical cues from the substrate are transduced into the cell through the formation of integrin-regulated focal adhesions and cytoskeletal reorganization, which, in turn, modulate cell biomechanics by downregulating cell stiffness. Surface nanotopography and consequent biomechanical response impact the overall behavior of macrophages by increasing movement and phagocytic ability without significantly influencing their inflammatory behavior. Our study suggests a strong potential of surface nanotopography for the regulation of macrophage functions, which implies a prospective application relative to coating technology for biomedical devices.

Cluster-Assembled Zirconia Substrates Accelerate the Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells.

Due to their high mechanical strength and good biocompatibility, nanostructured zirconia surfaces (ns-ZrOx) are widely used for bio-applications. Through supersonic cluster beam deposition, we produced ZrOx films with controllable roughness at the nanoscale, mimicking the morphological and topographical properties of the extracellular matrix. We show that a 20 nm ns-ZrOx surface accelerates the osteogenic differentiation of human bone marrow-derived MSCs (bMSCs) by increasing the deposition of calcium in the extracellular matrix and upregulating some osteogenic differentiation markers. bMSCs seeded on 20 nm ns-ZrOx show randomly oriented actin fibers, changes in nuclear morphology, and a reduction in mitochondrial transmembrane potential when compared to the cells cultured on flat zirconia (flat-ZrO2) substrates and glass coverslips used as controls. Additionally, an increase in ROS, known to promote osteogenesis, was detected after 24 h of culture on 20 nm ns-ZrOx. All the modifications induced by the ns-ZrOx surface are rescued after the first hours of culture. We propose that ns-ZrOx-induced cytoskeletal remodeling transmits signals generated by the extracellular environment to the nucleus, with the consequent modulation of the expression of genes controlling cell fate.

Nanotopography and Microconfinement Impact on Primary Hippocampal Astrocyte Morphology, Cytoskeleton and Spontaneous Calcium Wave Signalling.

Astrocytes' organisation affects the functioning and the fine morphology of the brain, both in physiological and pathological contexts. Although many aspects of their role have been characterised, their complex functions remain, to a certain extent, unclear with respect to their contribution to brain cell communication. Here, we studied the effects of nanotopography and microconfinement on primary hippocampal rat astrocytes. For this purpose, we fabricated nanostructured zirconia surfaces as homogenous substrates and as micrometric patterns, the latter produced by a combination of an additive nanofabrication and micropatterning technique. These engineered substrates reproduce both nanotopographical features and microscale geometries that astrocytes encounter in their natural environment, such as basement membrane topography, as well as blood vessels and axonal fibre topology. The impact of restrictive adhesion manifests in the modulation of several cellular properties of single cells (morphological and actin cytoskeletal changes) and the network organisation and functioning. Calcium wave signalling was observed only in astrocytes grown in confined geometries, with an activity enhancement in cells forming elongated agglomerates with dimensions typical of blood vessels or axon fibres. Our results suggest that calcium oscillation and wave propagation are closely related to astrocytic morphology and actin cytoskeleton organisation.

The glycocalyx affects the mechanotransductive perception of the topographical microenvironment.

The cell/microenvironment interface is the starting point of integrin-mediated mechanotransduction, but many details of mechanotransductive signal integration remain elusive due to the complexity of the involved (extra)cellular structures, such as the glycocalyx. We used nano-bio-interfaces reproducing the complex nanotopographical features of the extracellular matrix to analyse the glycocalyx impact on PC12 cell mechanosensing at the nanoscale (e.g., by force spectroscopy with functionalised probes). Our data demonstrates that the glycocalyx configuration affects spatio-temporal nanotopography-sensitive mechanotransductive events at the cell/microenvironment interface. Opposing effects of major glycocalyx removal were observed, when comparing flat and specific nanotopographical conditions. The excessive retrograde actin flow speed and force loading are strongly reduced on certain nanotopographies upon strong reduction of the native glycocalyx, while on the flat substrate we observe the opposite trend. Our results highlight the importance of the glycocalyx configuration in a molecular clutch force loading-dependent cellular mechanism for mechanosensing of microenvironmental nanotopographical features.

Micropatterning of Substrates for the Culture of Cell Networks by Stencil-Assisted Additive Nanofabrication.

The fabrication of in vitro neuronal cell networks where cells are chemically or electrically connected to form functional circuits with useful properties is of great interest. Standard cell culture substrates provide ensembles of cells that scarcely reproduce physiological structures since their spatial organization and connectivity cannot be controlled. Supersonic Cluster Beam Deposition (SCBD) has been used as an effective additive method for the large-scale fabrication of interfaces with extracellular matrix-mimicking surface nanotopography and reproducible morphological properties for cell culture. Due to the high collimation of SCBD, it is possible to exploit stencil masks for the fabrication of patterned films and reproduce features as small as tens of micrometers. Here, we present a protocol to fabricate micropatterned cell culture substrates based on the deposition of nanostructured cluster-assembled zirconia films by stencil-assisted SCBD. The effectiveness of this approach is demonstrated by the fabrication of micrometric patterns able to confine primary astrocytes. Calcium waves propagating in the astrocyte networks are shown.

Proteomic Analysis Reveals a Mitochondrial Remodeling of ßTC3 Cells in Response to Nanotopography.

Recently, using cluster-assembled zirconia substrates with tailored roughness produced by supersonic cluster beam deposition, we demonstrated that ß cells can sense nanoscale features of the substrate and can translate these stimuli into a mechanotransductive pathway capable of preserveing ß-cell differentiation and function in vitro in long-term cultures of human islets. Using the same proteomic approach, we now focused on the mitochondrial fraction of ßTC3 cells grown on the same zirconia substrates and characterized the morphological and proteomic modifications induced by the nanostructure. The results suggest that, in ßTC3 cells, mitochondria are perturbed by the nanotopography and activate a program involving metabolism modification and modulation of their interplay with other organelles. Data were confirmed in INS1E, a different ß-cell model. The change induced by the nanostructure can be pro-survival and prime mitochondria for a metabolic switch to match the new cell needs.

An Amperometric Sensor for Thiocholine Based on Cluster-Assembled Zirconia Modified Electrodes.

Here we report on the fabrication and characterization of cluster-assembled nanostructured zirconia electrodes for the electrochemical detection of enzymatically produced thiocholine. Zirconia nanostructures are produced by Supersonic Cluster Beam Deposition on thin gold films. This technique enables nanoscale control of the deposited film surface morphology, providing high active surface area for electrochemical detection of the analyte, along with high double-layer capacitance and suitable charge transfer resistance of the system. The electrochemical behavior of the electrodes has been characterized in the presence of the Potassium ferricyanide/Potassium ferrocyanide redox couple and the system performance showed to be enhanced starting from a thickness of the deposited layer of 60 nm. The electrochemical response for the oxidation of an enzymatic product was assessed by means of cyclic voltammetry, electrochemical impedance spectroscopy and chronoamperometry. The nanostructured zirconia film yields a good electrochemical detection of thiocholine. The limit of detection for thiocholine under working potential of 0.810-0.820 V versus reference was found to be comprised between 0.25 µM and 1.3 µM. Nanostructured electrodes, combining gold and zirconia nanoparticles can be implemented as functional transducers in biosensing devices, for example based on Acetylcholinesterase for electrochemical detection of polluting agents.

Neuronal Cells Confinement by Micropatterned Cluster-Assembled Dots with Mechanotransductive Nanotopography.

Artificially grown neuronal cultures of brain cells have been used for decades in the attempt to reproduce and study in vitro the complexity of brain circuits. It soon became evident that this alone was insufficient, because of the random architecture of these artificial networks. Important groundwork therefore resulted in the development of methods to confine neuronal adhesion at specific locations to match predefined network topologies and connectivity. Despite this notable progress in neural circuitry engineering, there is still need for micropatterned substrates that recapitulate better biophysical cues of the neuronal microenvironment, taking into account recent findings of their significance for neuronal differentiation and functioning. Here, we report the development and characterization of a novel approach that, by using supersonic cluster beam deposition of zirconia nanoparticles, allows the patterning of small nanostructured cell-adhesive areas according to predefined geometries onto elsewhere nonadhesive antifouling glass surfaces. As distinguishing features, compared to other micropatterning approaches in this context, the integrated nanostructured surfaces possess extracellular matrix-like nanotopographies of predetermined roughness; previously shown to be able to promote neuronal differentiation due to their impact on mechanotransductive processes, and can be used in their original state without any coating requirements. These micropatterned substrates were validated using (i) a neuron-like PC12 cell line and (ii) primary cultures of rat hippocampal neurons. After initial uniform plating, both neuronal cells types were found to converge and adhere specifically to the nanostructured regions. The cell-adhesive areas effectively confined cells, even when these were highly mobile and repeatedly attempted to cross boundaries. Inside these small permissive islands, cells grew and differentiated, in the case of the hippocampal neurons, up to the formation of mature, functionally active, and highly connected synaptic networks. In addition, when spontaneous instances of axon bridging between nearby dots occurred, a functional interdot communication between these subgroups of cells was observed.

Proteomic Dissection of Nanotopography-Sensitive Mechanotransductive Signaling Hubs that Foster Neuronal Differentiation in PC12 Cells.

Neuronal cells are competent in precisely sensing nanotopographical features of their microenvironment. The perceived microenvironmental information will be "interpreted" by mechanotransductive processes and impacts on neuronal functioning and differentiation. Attempts to influence neuronal differentiation by engineering substrates that mimic appropriate extracellular matrix (ECM) topographies are hampered by the fact that profound details of mechanosensing/-transduction complexity remain elusive. Introducing omics methods into these biomaterial approaches has the potential to provide a deeper insight into the molecular processes and signaling cascades underlying mechanosensing/-transduction but their exigence in cellular material is often opposed by technical limitations of major substrate top-down fabrication methods. Supersonic cluster beam deposition (SCBD) allows instead the bottom-up fabrication of nanostructured substrates over large areas characterized by a quantitatively controllable ECM-like nanoroughness that has been recently shown to foster neuron differentiation and maturation. Exploiting this capacity of SCBD, we challenged mechanosensing/-transduction and differentiative behavior of neuron-like PC12 cells with diverse nanotopographies and/or changes of their biomechanical status, and analyzed their phosphoproteomic profiles in these settings. Versatile proteins that can be associated to significant processes along the mechanotransductive signal sequence, i.e., cell/cell interaction, glycocalyx and ECM, membrane/f-actin linkage and integrin activation, cell/substrate interaction, integrin adhesion complex, actomyosin organization/cellular mechanics, nuclear organization, and transcriptional regulation, were affected. The phosphoproteomic data suggested furthermore an involvement of ILK, mTOR, Wnt, and calcium signaling in these nanotopography- and/or cell mechanics-related processes. Altogether, potential nanotopography-sensitive mechanotransductive signaling hubs participating in neuronal differentiation were dissected.

Scale Invariant Disordered Nanotopography Promotes Hippocampal Neuron Development and Maturation with Involvement of Mechanotransductive Pathways.

The identification of biomaterials which promote neuronal maturation up to the generation of integrated neural circuits is fundamental for modern neuroscience. The development of neural circuits arises from complex maturative processes regulated by poorly understood signaling events, often guided by the extracellular matrix (ECM). Here we report that nanostructured zirconia surfaces, produced by supersonic cluster beam deposition of zirconia nanoparticles and characterized by ECM-like nanotopographical features, can direct the maturation of neural networks. Hippocampal neurons cultured on such cluster-assembled surfaces displayed enhanced differentiation paralleled by functional changes. The latter was demonstrated by single-cell electrophysiology showing earlier action potential generation and increased spontaneous postsynaptic currents compared to the neurons grown on the featureless unnaturally flat standard control surfaces. Label-free shotgun proteomics broadly confirmed the functional changes and suggests furthermore a vast impact of the neuron/nanotopography interaction on mechanotransductive machinery components, known to control physiological in vivo ECM-regulated axon guidance and synaptic plasticity. Our results indicate a potential of cluster-assembled zirconia nanotopography exploitable for the creation of efficient neural tissue interfaces and cell culture devices promoting neurogenic events, but also for unveiling mechanotransductive aspects of neuronal development and maturation.

Conversion of nanoscale topographical information of cluster-assembled zirconia surfaces into mechanotransductive events promotes neuronal differentiation.

BACKGROUND: Thanks to mechanotransductive components cells are competent to perceive nanoscale topographical features of their environment and to convert the immanent information into corresponding physiological responses. Due to its complex configuration, unraveling the role of the extracellular matrix is particularly challenging. Cell substrates with simplified topographical cues, fabricated by top-down micro- and nanofabrication approaches, have been useful in order to identify basic principles. However, the underlying molecular mechanisms of this conversion remain only partially understood. RESULTS: Here we present the results of a broad, systematic and quantitative approach aimed at understanding how the surface nanoscale information is converted into cell response providing a profound causal link between mechanotransductive events, proceeding from the cell/nanostructure interface to the nucleus. We produced nanostructured ZrO2 substrates with disordered yet controlled topographic features by the bottom-up technique supersonic cluster beam deposition, i.e. the assembling of zirconia nanoparticles from the gas phase on a flat substrate through a supersonic expansion. We used PC12 cells, a well-established model in the context of neuronal differentiation. We found that the cell/nanotopography interaction enforces a nanoscopic architecture of the adhesion regions that affects the focal adhesion dynamics and the cytoskeletal organization, which thereby modulates the general biomechanical properties by decreasing the rigidity of the cell. The mechanotransduction impacts furthermore on transcription factors relevant for neuronal differentiation (e.g. CREB), and eventually the protein expression profile. Detailed proteomic data validated the observed differentiation. In particular, the abundance of proteins that are involved in adhesome and/or cytoskeletal organization is striking, and their up- or downregulation is in line with their demonstrated functions in neuronal differentiation processes. CONCLUSION: Our work provides a deep insight into the molecular mechanotransductive mechanisms that realize the conversion of the nanoscale topographical information of SCBD-fabricated surfaces into cellular responses, in this case neuronal differentiation. The results lay a profound cell biological foundation indicating the strong potential of these surfaces in promoting neuronal differentiation events which could be exploited for the development of prospective research and/or biomedical applications. These applications could be e.g. tools to study mechanotransductive processes, improved neural interfaces and circuits, or cell culture devices supporting neurogenic processes.