Marco Cecchini

Ultrastructural Characterization of the Lower Motor System in a Mouse Model of Krabbe Disease

Krabbe disease (KD) is a neurodegenerative disorder caused by the lack of β- galactosylceramidase enzymatic activity and by widespread accumulation of the cytotoxic galactosyl-sphingosine in neuronal, myelinating and endothelial cells. Despite the wide use of Twitcher mice as experimental model for KD, the ultrastructure of this model is partial and mainly addressing peripheral nerves. More details are requested to elucidate the basis of the motor defects, which are the first to appear during KD onset. Here we use transmission electron microscopy (TEM) to focus on the alterations produced by KD in the lower motor system at postnatal day 15 (P15), a nearly asymptomatic stage, and in the juvenile P30 mouse. We find mild effects on motorneuron soma, severe ones on sciatic nerves and very severe effects on nerve terminals and neuromuscular junctions at P30, with peripheral damage being already detectable at P15. Finally, we find that the gastrocnemius muscle undergoes atrophy and structural changes that are independent of denervation at P15. Our data further characterize the ultrastructural analysis of the KD mouse model, and support recent theories of a dying-back mechanism for neuronal degeneration, which is independent of demyelination.

Neuronal polarity selection by topography-induced focal adhesion control

Interaction between differentiating neurons and the extracellular environment guides the establishment of cell polarity during nervous system development. Developing neurons read the physical properties of the local substrate in a contact-dependent manner and retrieve essential guidance cues. In previous works we demonstrated that PC12 cell interaction with nanogratings (alternating lines of ridges and grooves of submicron size) promotes bipolarity and alignment to the substrate topography. Here, we investigate the role of focal adhesions, cell contractility, and actin dynamics in this process. Exploiting nanoimprint lithography techniques and a cyclic olefin copolymer, we engineered biocompatible nanostructured substrates designed for high-resolution live-cell microscopy. Our results reveal that neuronal polarization and contact guidance are based on a geometrical constraint of focal adhesions resulting in an angular modulation of their maturation and persistence. We report on ROCK1/2-myosin-II pathway activity and demonstrate that ROCK-mediated contractility contributes to polarity selection during neuronal differentiation. Importantly, the selection process confined the generation of actin-supported membrane protrusions and the initiation of new neurites at the poles. Maintenance of the established polarity was independent from NGF stimulation. Altogether our results imply that focal adhesions and cell contractility stably link the topographical configuration of the extracellular environment to a corresponding neuronal polarity state.

Nanotopographic Control of Neuronal Polarity

We employ simple geometrical rules to design a set of nanotopographies able to interfere with focal adhesion establishment during neuronal differentiation. Exploiting nanoimprint lithography techniques on cyclic-olefin-copolymer films, we demonstrate that by varying a single topographical parameter the orientation and maturation of focal adhesions can be finely modulated yielding independent control over the final number and the outgrowth direction of neurites. Taken together, this report provides a novel and promising approach to the rational design of biocompatible textured substrates for tissue engineering applications.

Acoustic-counterflow microfluidics by surface acoustic waves

In this letter, we demonstrate an unexpected surface-acoustic-wave (SAW)-driven pumping effect in hydrophobic polydimethilsiloxane (PDMS)-lithium niobate (LiNbO3) microchannels. Atomization within the fluidic channel followed by SAW-assisted coalescence leads to liquid counterflow with respect to the SAW propagation direction. This physical mechanism is contrasted with the acoustic-streaming process driving isolated drop displacement on piezoelectric substrates. This principle is shown not to be readily applicable to the present microchannel case. The proposed device geometry can be exploited to integrate micropumps into complex microfluidic chips, improving the portability of micro-total-analysis systems.

Nanoliter‐Droplet Acoustic Streaming via Ultra High Frequency Surface Acoustic Waves

The relevant length scales in sub-nanometer amplitude surface acoustic wave-driven acoustic streaming are demonstrated. We demonstrate the absence of any physical limitations preventing the downscaling of SAW-driven internal streaming to nanoliter microreactors and beyond by extending SAW microfluidics up to operating frequencies in the GHz range. This method is applied to nanoliter scale fluid mixing.

The effect of alternative neuronal differentiation pathways on PC12 cell adhesion and neurite alignment to nanogratings

During development and regeneration of the mammalian nervous system, directional signals guide differentiating neurons toward their targets. Soluble neurotrophic molecules encode for preferential direction over long distances while the local topography is read by cells in a process requiring the establishment of focal adhesions. The mutual interaction between overlapping molecular and topographical signals introduces an additional level of control to this picture. The role of the substrate topography was demonstrated exploiting nanotechnologies to generate biomimetic scaffolds that control both the polarity of differentiating neurons and the alignment of their neurites. Here PC12 cells contacting nanogratings made of copolymer 2-norbornene ethylene (COC), were alternatively stimulated with Nerve Growth Factor, Forskolin, and 8-(4-chloro-phenylthio)-2-O-methyladenosine-3,5-cyclic (8CPT-2Me-cAMP) or with a combination of them. Topographical guidance was differently modulated by the alternative stimulation protocols tested. Forskolin stimulation reduced the efficiency of neurite alignment to the nanogratings. This effect was linked to the inhibition of focal adhesion maturation. Modulation of neurite alignment and focal adhesion maturation upon Forskolin stimulation depended on the activation of the MEK/ERK signaling but were PkA independent. Altogether, our results demonstrate that topographical guidance in PC12 cells is modulated by the activation of alternative neuronal differentiation pathways.

Control of initial endothelial spreading by topographic activation of focal adhesion kinase

The time required to re-establish a functioning endothelial cell layer after vascular implant placement is critical to the success of the respective cardiologic or surgical intervention. Topographic modifications of implant surfaces promise to expedite endothelial regeneration by triggering the activation of cellular machineries that facilitate cell spreading. Exploiting nanoimprint lithography techniques on cyclic olefin copolymer foils, we engineered biocompatible submicron- and micro-structured gratings with groove and ridge width of 1 or 5 µm and groove depth ranging from 0.1 to 2 µm. Our results reveal that both the onset of endothelial spreading and subsequent texture-guided cell polarization critically depend on the feature size of the underlying topography, yet are independently modulated by the surface texture. Specifically, we demonstrate that on gratings with ridge and groove width of 1 µm and groove depth of 1 µm or deeper, the onset of endothelial spreading is 40% faster than on flat substrates, and that the cells align within ten degrees to the gratings. On this topography, we identify two independently regulated phases: acceleration of the onset of spreading supported by the rapid activation of integrin signaling proceeding viaFocal Adhesion Kinase, and contact guidance which requires ROCK1/2 and myosin-II dependent cell contractility and focal adhesion maturation.

Accelerated endothelial wound healing on microstructured substrates under flow

Understanding and accelerating the mechanisms of endothelial wound healing is of fundamental interest for biotechnology and of significant medical utility in repairing pathologic changes to the vasculature induced by invasive medical interventions. We report the fundamental mechanisms that determine the influence of substrate topography and flow on the efficiency of endothelial regeneration. We exposed endothelial monolayers, grown on topographically engineered substrates (gratings), to controlled levels of flow-induced shear stress. The wound healing dynamics were recorded and analyzed in various configurations, defined by the relative orientation of an inflicted wound, the topography and the flow direction. Under flow perpendicular to the wound, the speed of endothelial regeneration was significantly increased on substrates with gratings oriented in the direction of the flow when compared to flat substrates. This behavior is linked to the dynamic state of cell-to-cell adhesions in the monolayer. In particular, interactions with the substrate topography counteract Vascular Endothelial Cadherin phosphorylation induced by the flow and the wounding. This effect contributes to modulating the mechanical connection between migrating cells to an optimal level, increasing their coordination and resulting in coherent cell motility and preservation of the monolayer integrity, thus accelerating wound healing. We further demonstrate that the reduction of vascular endothelial cadherin phosphorylation, through specific inhibition of Src activity, enhances endothelial wound healing in flows over flat substrates.

PC12 differentiation on biopolymer nanostructures

The study of nervous system regeneration and axonal outgrowth control are relevant in several research areas, like neurophysiology or biomedical engineering. Among the elements that control neuron dynamics, the host substrate topography is a key parameter in determining cell differentiation. We present time-lapse experiments to analyze the differentiation dynamics of PC12 cells on nanopatterned biocompatible substrates. 200xa0nm depth gratings were fabricated on tissue-culture polystyrene substrates by nanoimprint lithography; different linewidths and pitches were compared down to 500xa0nm and 1000xa0nm, respectively. PC12 cells were cultured on these substrates and, following NGF administration to the medium, body morphology, cell movement and neuritogenesis were monitored at different time periods. In addition to demonstrating guided differentiation, our studies show complex time variations in body morphology and axon length, and guided cell movement. We show unstable synaptic connections and cell-body polarization, and the competition between topographical guidance and cell–cell interactions.

Neuronal differentiation on anisotropic substrates and the influence of nanotopographical noise on neurite contact guidance

Cells are exposed to specific directional physical signals determined by the micro/nano-environment that in vivo coexist with some degree of topographical noise. Particularly in the nervous system, cell contact sensing of the extracellular environment plays a primary role in defining neurite initiation and final brain wiring. Here we study neuronal cell response to directional stimuli by exploiting nanogratings with controlled amount of random nanotopographical noise. The impact of noise on neurite guidance and focal adhesions (FAs) development is investigated in NGF-differentiating PC12 cells by confocal and TIRF microscopy. We show that the loss of neurite guidance is not linear with noise, but is a threshold effect, correlating with changes in FA maturation and spatial organization. Finally nocodazole, a drug that increases cell contractility, can improve neurite alignment by promoting aligned FA maturation. We argue that these results show new possibilities for successful implant strategies particularly in the context of nerve-regeneration devices.