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Cluster-assembled nanostructured materials for cell biology
Abstract
In their physiological microenvironment cells interact with the extracellular matrix (ECM); a complex structure build up by intertwined nanometric components (proteins and sugar chains). In the last two decades it became increasingly evident that the topographical complexity of the ECM encountered by cells at the nanoscale decisively influences cell behavior and fate through an intricate signaling pathway called mechanotransduction, i.e., the conversion of microenvironmental biophysical and structural cues into corresponding cellular responses. Classical cell culture devices and experimentation did not sufficiently take into account this aspect, and there is a need in cell biological research for substrates that mimic accurately the ECM nanotopography. This chapter introduces the molecular mechanisms by which cells perceive and “interpret” nanotopographical ECM cues and outlines why cluster-assembled nanostructured materials (produced by supersonic cluster beam deposition) are useful in this context as substrates for mechanobiological studies and bioengineering applications by providing reproducible ECM-like nanotopographical features.
... Page 3 of 20 Chighizola et al. Journal of Nanobiotechnology (2022) 20:418 processes [9,10,13,27], as well as cellular functioning, programme and differentiation in various cell biological contexts [9,13,[30][31][32][33], has already been documented. ...
... Clusters are collected directly on the CPs, or glass substrates, intercepting the beam in the deposition chamber. Upon landing on the sample surfaces, clusters form a nanostructured, highly porous, high-specific area, biocompatible ns-ZrO 2 film [27,32]. The protocol for the functionalisation of the colloidal probes with the nanotopographical surfaces achieved by zirconia cluster-assembling is detailed in Chighizola et al. [16]. ...
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.
Graphical Abstract
... In this framework, we use a bottom-up nanofabrication technique, called supersonic cluster beam deposition (SCBD) 25,26 , to mimic ECM nanotopographies (assembled by clusters of zirconia nanoparticles). These nano-bio interfaces possess the potential to modulate mechanotransductive processes 9,10,13,26 , as well as cellular functioning, programme and differentiation in various cell biological contexts 9,13,27,28,29,30 . Recently, we have furthermore developed an approach to test integrin-related nanoscale mechanotransductive processes, by means of atomic force microscopy (AFM)-based adhesion force spectroscopy measurements using custom nanostructured colloidal probes 19 (Fig. 1B). ...
... Clusters are collected directly on the CPs, or glass substrates, intercepting the beam in the expansion chamber. Upon landing on the sample surfaces, clusters form a nanostructured, highly porous, high-specific area, biocompatible ns-ZrO 2 film 26,29 . The protocol for the functionalisation of the colloidal probes with the nanotopographical surfaces achieved by zirconia cluster-assembling is detailed in Chighizola et al. 19 . ...
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 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 removal 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.
... multicellular organisms are embedded in tissues composed of other cells or extracellular matrices with well-defined elastic moduli [62]. The composition of ECM defines its biophysical configuration determining specific mechanical and structural properties [63,64]. Cells mechanical properties and cytoskeleton reorganization on hard and flat substrates (such as glass/ plastic petri dish) are different from cells in natural micro environment having peculiar roughness and elasticity. ...
Nanomechanics of cytoskeleton is deeply involved in physiology and regulation of cell behavior. Atomic Force Microscopy has been extensively used for quantitative characterization with high-spatial resolution, in particular showing tremendous opportunities in biomechanics by quantifying mechanical parameters related to cytoskeleton organization. In this short review, we highlight recent developments in cell nanomechanics by AFM focusing on methodology and direct application to investigate cytoskeleton restructuration when cells are interacting with nanostructures (surfaces and nanoparticles). In particular, cells can sense the stiffness of environment or internalized particles and AFM can detect the rearrangement of cytoskeleton as one of the responses of mechanotransduction stimuli. Current bottlenecks hindering further progress in technology, such as theoretical models of interpretation will be discussed, in particular we propose a solution for complex system by coupling AFM with finite element simulations to retrieve more quantitative information when heterogeneity and convolution play important roles. Finally, we present recent cutting-edge research directions to explore new techniques and enhance the capabilities of AFM nanomechanics for living cells.
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 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.
A characteristic of integrins is their ability to transfer chemical and mechanical signals across the plasma membrane. Force generated by myosin II makes cells able to sense substrate stiffness and induce maturation of nascent adhesions into focal adhesions. In this paper, we present a comprehensive proteomic analysis of nascent and mature adhesions. The purification of integrin adhesion complexes combined with quantitative mass spectrometry enabled the identification and quantification of known and new adhesion-associated proteins. Furthermore, blocking adhesion maturation with the myosin II inhibitor blebbistatin markedly impaired the recruitment of LIM domain proteins to integrin adhesion sites. This suggests a common recruitment mechanism for a whole class of adhesion-associated proteins, involving myosin II and the zinc-finger-type LIM domain.