Bistability of Dielectrically Anisotropic Nematic Crystals and the Adaptation of Endothelial Collectives to Stress Fields.

Endothelial monolayers physiologically adapt to flow and flow-induced wall shear stress, attaining ordered configurations in which elongation, orientation, and polarization are coherently organized over many cells. Here, with the flow direction unchanged, a peculiar bi-stable (along the flow direction or perpendicular to it) cell alignment is observed, emerging as a function of the flow intensity alone, while cell polarization is purely instructed by flow directionality. Driven by the experimental findings, the parallelism between endothelia is delineated under a flow field and the transition of dual-frequency nematic liquid crystals under an external oscillatory electric field. The resulting physical model reproduces the two stable configurations and the energy landscape of the corresponding system transitions. In addition, it reveals the existence of a disordered, metastable state emerging upon system perturbation. This intermediate state, experimentally demonstrated in endothelial monolayers, is shown to expose the cellular system to a weakening of cell-to-cell junctions to the detriment of the monolayer integrity. The flow-adaptation of monolayers composed of healthy and senescent endothelia is successfully predicted by the model with adjustable nematic parameters. These results may help to understand the maladaptive response of in vivo endothelial tissues to disturbed hemodynamics and the progressive functional decay of senescent endothelia.

A Novel Hybrid Membrane VAD as First Step Toward Hemocompatible Blood Propulsion.

Heart failure is a raising cause of mortality. Heart transplantation and ventricular assist device (VAD) support represent the only available lifelines for end stage disease. In the context of donor organ shortage, the future role of VAD as destination therapy is emerging. Yet, major drawbacks are connected to the long-term implantation of current devices. Poor VAD hemocompatibility exposes the patient to life-threatening events, including haemorrhagic syndromes and thrombosis. Here, we introduce a new concept of artificial support, the Hybrid Membrane VAD, as a first-of-its-kind pump prototype enabling physiological blood propulsion through the cyclic actuation of a hyperelastic membrane, enabling the protection from the thrombogenic interaction between blood and the implant materials. The centre of the luminal membrane surface displays a rationally-developed surface topography interfering with flow to support a living endothelium. The precast cell layer survives to a range of dynamically changing pump actuating conditions i.e., actuation frequency from 1 to 4 Hz, stroke volume from 12 to 30 mL, and support duration up to 313 min, which are tested both in vitro and in vivo, ensuring the full retention of tissue integrity and connectivity under challenging conditions. In summary, the presented results constitute a proof of principle for the Hybrid Membrane VAD concept and represent the basis for its future development towards clinical validation.

Honeycomb-structured metasurfaces for the adaptive nesting of endothelial cells under hemodynamic loads.

The thrombogenicity of artificial materials comprising ventricular assist devices (VADs) limits their long-term integration in the human body. A living endothelium covering the luminal surface can provide a safe interface working compatibly with blood and circumventing this problem. However, the survival of endothelial cells is endangered by non-physiological hemodynamic conditions generated by VAD function, including high wall shear stress and deformation. Here, we introduce a surface topography comprising hexagonal honeycomb shelters in which cells remodel to generate coherently organized patterns of subcellular compartments. The resulting hexagonal array shows resistance to supraphysiological loads maintaining endothelium integrity and avoiding local discontinuities.

Cell cycle-dependent force transmission in cancer cells.

The generation of traction forces and their transmission to the extracellular environment supports the disseminative migration of cells from a primary tumor. In cancer cells, the periodic variation of nuclear stiffness during the cell cycle provides a functional link between efficient translocation and proliferation. However, the mechanical framework completing this picture remains unexplored. Here, the Fucci2 reporter was expressed in various human epithelial cancer cells to resolve their cell cycle phase transition. The corresponding tractions were captured by a recently developed reference-free confocal traction-force microscopy platform. The combined approach was conducive to the analysis of phase-dependent force variation at the level of individual integrin contacts. Detected forces were invariably higher in the G1 and early S phases than in the ensuing late S/G2, and locally colocalized with high levels of paxillin phosphorylation. Perturbation of paxillin phosphorylation at focal adhesions, obtained through the biochemical inhibition of focal adhesion kinase (FAK) or the transfection of nonphosphorylatable or phosphomimetic paxillin mutants, significantly diminished the force transmitted to the substrate. These data demonstrate a reproducible modulation of force transmission during the cell cycle progression of cancer cells, instrumental to their invasion of dense environments. In addition, they delineate a model in which paxillin phosphorylation supports the mechanical maturation of adhesions relaying forces to the substrate.

Pore Shape Defines Paths of Metastatic Cell Migration.

Invasion of dense tissues by cancer cells involves the interplay between the penetration resistance offered by interstitial pores and the deformability of cells. Metastatic cancer cells find optimal paths of minimal resistance through an adaptive path-finding process, which leads to successful dissemination. The physical limit of nuclear deformation is related to the minimal cross section of pores that can be successfully penetrated. However, this single biophysical parameter does not fully describe the architectural complexity of tissues featuring pores of variable area and shape. Here, employing laser nanolithography, we fabricate pore microenvironment models with well-controlled pore shapes, through which human breast cells (MCF10A) and their metastatic offspring (MCF10CA1a.cl1) could pervade. In these experimental settings, we demonstrate that the actual pore shape, and not only the cross section, is a major and independent determinant of cancer penetration efficiency. In complex architectures containing pores demanding large deformations from invading cells, tall and narrow rectangular openings facilitate cancer migration. In addition, we highlight the characteristic traits of the explorative behavior enabling metastatic cells to identify and select such pore shapes in a complex multishape pore environment, pinpointing paths of least resistance to invasion.

Facile endothelium protection from TNF-α inflammatory insult with surface topography.

Adverse events triggered by the direct contact between blood and synthetic materials constitute a sincere shortcoming of cardiovascular implant technology. A well-connected autologous endothelium, generated through the process of endothelialization, impedes such interaction and endows the implant luminal interface with optimal protection. The endothelialization of artificial substrates is the result of a complex interplay between endothelial cells (ECs), surface topography, and flow-generated wall shear stress (WSS). This is however tainted by the pro-inflammatory signaling, typical of cardiovascular patients, which compromises endothelial integrity and survival. Here, we challenge human endothelial monolayers with the pro-inflammatory factor TNF-α under realistic WSS conditions. In these experimental settings we demonstrate that the simple contact between ECs and an optimized surface geometry can inhibit NF-kB activation downstream of TNF-α yielding increased stability of VE-Cadherin mediated cell-to-cell junctions and of focal adhesions. Therefore the here-presented topographic modification can be implemented on a range of artificial substrates enabling their endothelialization under supra-physiological flow and in the presence of pro-inflammatory insults. These new findings constitute an important step toward achieving the full hemocompatibility of cardiovascular implants.

Endothelialization of Rationally Microtextured Surfaces with Minimal Cell Seeding Under Flow.

The generation of a confluent and functional endothelium at the luminal surface of cardiovascular devices represents the ideal solution to avoid contact between blood and synthetic materials thus allowing the long-term body integration of the implants. Due to the foreseen paucity of source cells in cardiovascular patients, surface engineering strategies to achieve full endothelialization, while minimizing the amount of endothelial cells required to seed the surface leading to prompt and full coverage with an endothelium are necessary. A stable endothelialization is the result of the interplay between endothelial cells, the flow-generated walls shear stress and the substrate topography. Here a novel strategy is designed and validated based on the use of engineered surface textures combined with confined islands of seeded endothelial cells. Upon release of the confinement, the cell island populations are able to migrate on the texture and merge under physiological flow conditions to promptly generate a fully connected endothelium. The interaction between endothelial cells and surface textures supports the process of endothelialization through the stabilization of cell-to-substrate adhesions and cell-to-cell junctions. It is shown that with this approach, when ≈50% of a textured surface is initially covered with cell seeding, the time to full endothelialization compared to an untextured surface is almost halved, underpinning the viability and effectiveness of the method for the quick and stable coverage of cardiovascular implants.